Pharmaceutical compositions having appetite suppressant activity

ABSTRACT

A pharmaceutical composition contains an extract obtainable from a plant of the genus  Trichocaulon  or  Hoodia  containing an appetite suppressant agent having the formula (1). A process for obtaining the extract and a process for synthesizing compound (1) and its analogues and derivatives is also provided. The invention also extends to the use of such extracts and compound (1) and its analogues for the manufacture of medicaments having appetite suppressant activity. The invention further provides novel intermediates for the synthesis of compound (1).

[0001] THIS INVENTION relates to steroidal glycosides, to compositionscontaining such steroidal glycosides and to a new use for thesesteroidal glycosides and the compositions containing them. The inventionfurther relates to a method of extracting and isolating these steroidalglycosides from plant material, to a method of synthetically producingthese steroidal glycosides, and to the products of such an extractionand such a synthesis process.

[0002] In a particular application, the invention relates to an appetitesuppressant agent, to a process for synthetically producing the appetitesuppressant agent, to a process for extracting the appetite suppressantagent from plant material, to an appetite suppressant compositioncontaining the appetite suppressant agent, and to a method ofsuppressing an appetite.

[0003] According to the invention, there is provided a process forpreparing an extract of a plant of the genus Trichocaulon or of thegenus Hoodia, the extract comprising an appetite suppressant agent, theprocess including the steps of treating collected plant material with asolvent to extract a fraction having appetite suppressant activity,separating the extraction solution from the rest of the plant material,removing the solvent from the extraction solution and recovering theextract. The extract so recovered may be further purified, eg by way ofsuitable solvent extraction procedures.

[0004] The invention also provides a plant extract made of plants of thegroup comprising the genus Trichocaulon and the genus Hoodia and havingappetite suppressant activity.

[0005] The extract may be prepared from plant material such as the stemsand roots of said plants of the genus Trichocaulon or of the genusHoodia. The genus Trichocaulon and the genus Hoodia include succulentplants growing in arid regions such as are found in Southern Africa. Inone application of the invention, the active appetite suppressantextract is obtained from the species Trichocaulon piliferum. The speciesTrichocaulon officinale may also be used to provide an active appetitesuppressant extract. In another application of the invention, the activeappetite suppressant extract may be obtained from the species Hoodiacurrorii, Hoodia gordonii or Hoodia lugardii. Bioassays conducted by theApplicant on rats have indicated that certain of the extracts possessappetite suppressant activity.

[0006] The plant material may be homogenised in the presence of asuitable solvent, for example, a methanol/methylene chloride solvent, bymeans of a device such as a Waring blender. The extraction solution maythen be separated from the residual plant material by an appropriateseparation procedure such as, for example, filtration or centrifugation.The solvent may be removed by means of the rotary evaporator, preferablyin a water bath at a temperature of 60° C. The separated crude extractmay then be further extracted with methylene chloride and water beforebeing separated into a methylene chloride extract and a water extract.The methylene chloride extract may have the solvent removed preferablyby means of evaporation on a rotary evaporator and the resultant extractmay be further purified by way of a methanol/hexane extraction. Themethanol/hexane extraction product may then be separated to yield amethanol extract and a hexane extract. The methanol extract may beevaporated to remove the solvent in order to yield a partially purifiedactive extract.

[0007] The partially purified active extract may be dissolved inmethanol, and may be further fractionated by column chromatography,employing silica gel as an adsorption medium and a chloroform/30methanolmixture as an eluent. A plurality of different fractions may beobtained, and each may be evaluated, by suitable bioassaying procedures,to determine the appetite suppressant activity thereof.

[0008] A fraction having appetite suppressant activity may preferably befurther fractionated such as by column chromatography using silica gelas an adsorption medium and a 9:1 chloroform:methanol solvent, and theresultant sub-fractions bioassayed for their appetite suppressantactivity. A sub-fraction displaying appetite suppressant activity may,if desired, be further fractionated and purified, conveniently using acolumn chromatographic procedure with silica gel as the adsorptionmedium and a 9:1 ethylacetate:hexane solvent. The resultant purified isfractions may again be evaluated by suitable bioassay procedures fortheir appetite suppressant activity.

[0009] The Applicant has found that at least one such purified fractionhas good appetite suppressant activity, and the active principle in thefraction was identified by conventional chemical techniques includingnuclear magnetic resonance, and was found to be a compound of thestructural formula

[0010] In accordance with S.I. nomenclature, the active principle (1) isthe compound3-0-[-β-D-thevetopyranosyl(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl]-12β-0-tigloyloxy-14-hydroxy-14β-pregn-50-en-20-one(C₄₇H₇₄O₁₅M⁺878).

[0011] According to another aspect of the invention, there is provided aprocess for preparing an extract of a plant of the genus Trichocaulon orof the genus Hoodia, the extract comprising an appetite suppressantagent, the process including the steps of pressing collected plantmaterial to separate sap from solid plant material and recovering thesap free of the solid plant material to form the extract.

[0012] The extract may be dried to remove moisture, e.g. byspray-drying, freeze-drying or vacuum drying, to form a free-flowingpowder.

[0013] The invention extends to a composition having appetitesuppressant activity comprising an extract as described above.

[0014] The composition may be admixed with a pharmaceutical excipient,diluent or carrier and optionally it is prepared in unit dosage form.

[0015] The invention also extends to the use of an extract as describedabove in the manufacture of a medicament having appetite suppressantactivity, to an extract as described above for use as a medicamenthaving appetite suppressant activity, and to a method of suppressing anappetite by administering to a human or animal an effective dosage of acomposition as described above.

[0016] Compound (1) is a novel compound and the invention extends tocompound (1) and certain analogues or derivatives of this steroidaltrisaccharide having appetite suppressant properties. The moleculeschosen as the analogues or derivatives are intended to affect theproperties of the steroidal trisaccharide with the aim of increasing theactivity of the active ingredient. The following effects were taken intoconsideration when the analogues were chosen:

[0017] (i) Hydrophobic interactions and lipophilicity

[0018] Functional group modifications of the active molecule is intendedto change the hydrophobicity and lipophilicity of the molecule.Increased lipophilicity has been shown to correlate with increasedbiological activity, poorer aqueous solubility, increaseddetergency/cell lysis, increased storage in tissues, more rapidmetabolism and elimination, increased plasma protein binding and fasterrate of onset of action.

[0019] (ii) Electronic properties and ionization constants

[0020] Functional group modification of the molecule is also intended tochange the acidity and basicity which would have a major role incontrolling the transport of the compound to its site of action and thebinding at this target site.

[0021] (iii) Hydrogen bonding

[0022] Functional group modifications of carboxyl and carbonyl groups inthe active molecule are intended to change the interactions between theproteins in biological systems and the chemically modified functionalgroups.

[0023] (iv) Steric parameters

[0024] The purpose of changing the steric features of the molecule is toincrease binding to its receptor and thus increase its biologicalactivity.

[0025] The following chemical modifications to the molecule are intendedto affect the hydrophobicity and lipophilicity electronic properties,hydrogen bonding and steric parameters on the molecule:

[0026] a) Chemical modification of the C-12 group and esterfunctionality;

[0027] b) Chemical modification of the 5,6-double bond, e.g.hydrogenation and migration;

[0028] c) Chemical modification of the C-20 carbonyl and C-17 acetylgroup;

[0029] d) Chemical modification of the “D” ring of the steroid oraglycone ring;

[0030] e) Modification of the carbohydrates of the trisaccharide moiety.

[0031] Accordingly, the invention provides a compound having the generalstructural formula

[0032] in which R=alkyl;

[0033] R₁=H, alkyl, tigloyl, benzoyl, or any other organic ester group;

[0034] R₂=H, or one or more 6-deoxy carbohydrates, or one or more2,6-dideoxy carbohydrates, or glucose molecules, or combinationsthereof;

[0035] and in which the broken lines indicate the optional presence of afurther bond between C4-C5 or C5-C6.

[0036] The invention also provides a compound as described above whereinthere is a further bond between C5-C6, R=methyl, R₁=tigloyl,R₂=3-0-([-β-D-thevetopyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl]and having the structural formula.

[0037] Further active analogues or derivatives of the appetitesuppressant compound (1) in accordance with the invention are compoundshaving the following structural formulae:

[0038] in which R=alkyl; and

[0039] R₁=H, or benzoyl, or tigloyl, or any other organic ester group

[0040] in which R=alkyl; and

[0041] R₁=H, or tigloyl, or benzoyl, or any other organic ester group

[0042] in which R=alkyl; and

[0043] R₁=H, or tigloyl, or benzoyl, or any other organic ester group

[0044] in which R=alkyl; and

[0045] R₁=H, or tigloyl, or benzoyl, or any other organic ester group

[0046] in which R=alkyl;

[0047] R₁=H, or tigloyl, or benzoyl, or any other organic ester group.

[0048] in which R=alkyl; and

[0049] R₁=H, alkyl, tigloyl, benzoyl, or any other organic ester group;

[0050] R₂=H, or one or more 6-deoxy carbohydrates, or one or more2,6-dideoxy carbohydrates, or glucose molecules, or combinationsthereof; and in which the broken lines indicate the optional presence ofa further bond between C4-C5 or C5-C6.

[0051] in which R=alkyl; and

[0052] R₁=H, alkyl, tigloyl, benzoyl, or any other organic ester group;

[0053] R₂=H, or one or more 6-deoxy carbohydrates, or one or more2,6-dideoxy carbohydrates, or glucose molecules, or combinationsthereof;

[0054] and in which the broken lines indicate the presence of a furtherbond between C4-C5 or C5-C6.

[0055] in which R=alkyl; and

[0056] R₁=H, alkyl, tigloyl, benzoyl, or any other organic ester group;

[0057] R₂=H, or one or more 6-deoxy carbohydrates, or one or more2,6-dideoxy carbohydrates, or glucose molecules, or combinationsthereof;

[0058] and in which the broken lines indicate the optional presence of afurther bond between C4-C5 or C5-C6.

[0059] in which R=alkyl; and

[0060] R₁=H, alkyl, tigloyl, benzoyl, or any other organic ester group;

[0061] R₂=H, or one or more 6-deoxy carbohydrates, or one or more2,6-dideoxy carbohydrates, or glucose molecules, or combinationsthereof; and in which the broken lines indicate the optional presence ofa further bond between C4-C5, C5-C6 or C14-C15.

[0062] in which R=alkyl; and

[0063] R₁=H, alkyl, tigloyl, benzoyl, any other organic ester group;

[0064] R₂=H, or one or more 6-deoxy carbohydrates, or one or more2,6-dideoxy carbohydrates, or glucose molecules, or combinationsthereof;

[0065] and in which the broken lines indicate the optional presence of afurther bond between C4-C5, C5-C6 or C14-C15.

[0066] in which R=alkyl; and

[0067] R₁=H, alkyl, tigloyl, benzoyl, any other organic ester group;

[0068] R₂=H, or one or more 6-deoxy carbohydrates, or one or more2,6-dideoxy carbohydrates, or glucose molecules, or combinationsthereof;

[0069] and in which the broken lines indicate the optional presence of afurther bond between C4-C5, C5-C6 or C14-C15; and

[0070] R₃=H, alkyl, aryl, acyl, or glucoxy.

[0071] in which R=H, alkyl, aryl or any steroid possessing a C14 betahydroxy group, or a C12 beta hydroxy functionality, or a C17 acyl group,or a C5-C6 olefin, or combinations thereof.

[0072] The invention still further extends to a process forsynthetically producing a compound having appetite suppressant activity.

[0073] The process uses a steroid as a starting material (orintermediate or precursor) the steroid having the chemical formula

[0074] The steroid (15) can be prepared from a compound having theformula (22) by a process which includes the steps of

[0075] (i) treating progesterone having the formula

[0076] with the micro-organism Calonectria decora to produce a compound12β, 15α-dihydroxy progesterone of the formula

[0077] (ii) treating compound 17) with tosyl chloride and pyridine toproduce a compound 12β-hydroxy-15α-(p-toluene sulfonyl)-progesterone ofthe formula

[0078] (iii) treating the compound (18) with collidine at 150° C. toproduce a compound 12β-hydroxy-Δ¹⁴-progesterone of the formula

[0079] (iv) treating the compound (19) with acetyl chloride and aceticanhydride at 120° C., to produce a compound3,12β-diacetoxypregna-3,5,14-trien-20-one of the formula

[0080] (v) treating the compound (20) with ethylene glycol and acatalytic amount of p-toluene sulphonic acid, to produce a compound3,12β-diacetoxy-20,20-ethylenedioxypregna-3,5,14-triene of the formula

[0081] (vi) treating the compound (21) with NaBH₄ to produce a compound3β,12β-dihydroxy-20,20-ethylenedioxypregna-5,14-diene-12-acetate of theformula

[0082] In a first alternative procedure, a process for the preparationof steroid (15) according to the invention includes the steps of

[0083] (a) treating compound (22) with a reducing agent, e.g.

[0084] LiAlH₄, to produce a compound 3β,12β-dihydroxy-20,20-ethylenedioxypregna-5, 14-diene of the formula

[0085] (b) treating compound (23) with N-bromoacetamide (NBA) and abase, e.g. pyridine, to produce a compound 3β, 12β-dihydroxy-14,15-epoxy-20,20-ethylenedioxypregn-5-ene of the formula

[0086] (c) treating compound (24) with a reducing agent, e.g. LiAlH₄,e.g. with refluxing, to produce a compound 3β, 12β,14β-trihydroxy-20,20-ethylenedioxypregn-5-ene of the formula

[0087] and (d) treating compound (25) with an acid, e.g. acetic acid,and water to produce the steroid intermediate compound 3β, 12β,14β-trihydroxy-pregn-5-ene (15).

[0088] Reaction Scheme A depicts the procedure for the preparation ofsteroid intermediate (15) from compound (22) according to “the firstalternative procedure” of the invention (and includes the preparation ofcompound (22) from compound (16) for illustrative purposes).

[0089] In a second alternative procedure, a process for the preparationof steroid (15) according to the invention includes the steps of

[0090] (a) treating compound (22) (3β,12β-dihydroxy-20,20-ethylenedioxypregna-5,14-diene-12-acetate) withp-toluenesulfonyl chloride and a base, e.g. pyridine, to produce acompound3β,12β-dihydroxy-20,20-ethylenedioxypregna-5,14-diene-3-tosyl-12-acetateof the formula

[0091] (b) treating compound (26) with potassium acetate in a solvent,e.g. acetone, to produce a compound 6β,12β-dihydroxy-20,20-ethylenedioxy-3,5α-cyclopregnan-14-ene-12-acetate ofthe formula

[0092] (c) treating the compound (27) with a reducing agent, e.g.LiAlH₄, and e.g. tetrahydrofuran, to produce a compound 6β,12β-dihydroxy-20,20-ethylenedioxy-3,5α-cyclopregnan-14-ene of theformula

[0093] (d) treating the compound (28) with N-bromoacetamide, optionallyacetic acid, and a base, e.g. pyridine, to produce a compound 6β,12β-dihydroxy-20,20-ethylenedioxy-14,15-epoxy-3,5α-cyclopregnane of theformula

[0094] (e) treating the compound (29) with a reducing agent, e.g.LiAlH₄, and e.g. tetrahydrofuran, to produce a compound 6β, 12β,14β-trihydroxy-20,20-ethylenedioxy-3,5α-cyclopregnane of the formula

[0095] and (f) treating compound (30) with an acid, e.g. hydrochloricacid, and a solvent e.g. acetone, to produce compound (15).

[0096] Reaction Scheme B shows the procedure for the preparation ofsteroid intermediate (15) from compound (22) according to “the secondalternative procedure” of the invention.

[0097] Compound (1) may be synthesized from a first carbohydrateintermediate in the form of an activated monosaccharide cymarose moiety,which can be prepared from a compound having the formula (36). Compound(36) can be prepared by a process which includes the steps

[0098] (i) treating methyl-α-D-glucose having the formula

[0099] with benzaldehyde and zinc chloride to produce a compoundmethyl-4,6-0-benzylidene-α-D-glucopyranoside of the formula

[0100] (ii) treating the compound (32) with tosyl chloride and pyridineat 0° C., to produce a compoundmethyl-4,6-0-benzylidene-2-0-tosyl-α-D-glucopyranoside of the formula

[0101] (iii) treating the compound (33) with NaOMe at 100° C. to producea compound methyl 4,6-0-benzylidene-3-0-methyl-α-D-altropyranoside ofthe formula

[0102] (iv) treating the compound (34) with N-bromosuccinamide (NBS) toproduce a compound methyl6-bromo-4-0-benzoyl-3-0-methyl-6-deoxy-α-D-altropyranoside of theformula

[0103] and (v) treating the compound (35) with NaBH₄ and NiCl₂, toproduce a compound methyl4-0-benzoyl-3-0-methyl-6-deoxy-α-D-altropyranoside of the formula

[0104] The invention extends to a process for the preparation of acarbohydrate intermediate in the form of an activated monosaccharidecymarose moiety which includes the steps of

[0105] (i) treating the compound (36) with PhSSiMe₃, ZnI₂ and Bu4⁺I⁻ toproduce a compound4-0-benzoyl-3-0-methyl-6-deoxy-αβ-D-phenylthioaltroside of the formula

[0106] (ii) optionally treating the compound (37) withdiethylaminosulphur trifluoride (DAST), e.g. at 0° C., to produce acompound4-0-benzoyl-3-0-methyl-2-phenylthio-2,6-dideoxy-αβ-D-fluorocymaropyranosidehaving the formula

[0107] or (iii) optionally, treating the compound (37) witht-butyldimethylsilylchloride and imidazole in a solvent, e.g. pyridine,to produce4-0-benzoyl-3-0-methyl-2-0-t-butyldimethylsilyl-αβ-D-phenylthioaltrosidehaving the formula

[0108] in which Z=TBDMS=t-butyldimethylsilyl

[0109] and (iv) treating the compound (39) with a base, e.g. sodiummethoxide, to produce3-0-methyl-2-0-t-butyldimethylsilyl-αβ-D-phenylthioaltroside having theformula

[0110] in which Z=TBDMS=t-butyldimethylsilyl.

[0111] Reaction Scheme C shows the procedure for the synthesis of theactivated monosaccharide cymarose moiety (40) from compound (36)according to the invention (and includes the preparation of compound(36) from compound (31) for illustrative purposes).

[0112] The synthesis of compound (1) may also involve a secondcarbohydrate intermediate in the form of an activated monosaccharidethevetose moiety, which can be prepared from a compound having theformula (47). Compound (47) can be prepared by a process which includesthe steps of

[0113] (i) treating α-D-glucose having the formula

[0114] with acetone and sulphuric acid to produce a compound 1,2:5,6-di-0-isopropylidene-α-D-glucofuranose of the formula

[0115] (ii) treating the compound (42) with NaH and MeI to produce acompound 1,2:5,6-Di-0-isopropylidene-3-0-methyl-α-D-glucofuranose of theformula

[0116] (iii) treating the compound (43) with acetic acid to produce acompound 3-0-methyl-αβ-D-glucopyranose of the formula

[0117] (iv) treating the compound (44) with methanol and hydrochloricacid to produce a compound methyl 3-0-methyl-αβ-D-glucopyranoside havingthe formula

[0118] (v) treating the compound (45) with benzaldehyde and zincchloride to produce a compound methyl4,6-0-benzylidene-3-0-methyl-αβ-glucopyranoside having the formula

[0119] (vi) treating the compound (46) with N-bromosuccinamide, nickelchloride and sodium borohydride to produce a compound methyl4-0-benzoyl-3-0-methyl-6-deoxy-αβ-glucopyranoside having the formula

[0120] The invention extends to a process for the preparation of anactivated monosaccharide thevetose moiety which includes the steps of

[0121] (i) treating the compound (47) with phenylthiotrimethysilane andtrimethylsilyltrifluoromethanesulphonate to produce a compound4-0-benzoyl-3-0-methyl-1-phenylthio-6-deoxy-αβ-glucopyranoside havingthe formula

[0122] (ii) treating the compound (48) with pivaloyl chloride and asolvent, e.g. pyridine, to produce a compound4-0-benzoyl-3-0-methyl-2-0-pivaloyl-1-phenylthio-6-deoxy-αβ-glucopyranosidehaving the formula

[0123] and (iii) treating the compound (49) with a brominating agent,e.g. N-bromosuccinimide, and diethylaminosulphur trifluoride to producea compound4-0-benzoyl-3-0-methyl-2-0-pivaloyl-1-fluoro-6-deoxy-β-glucopyranosideoccurring as stereo-isomers having the formula

[0124] Reaction Scheme D shows the procedure for the synthesis of theactivated monosaccharide thevetose moiety (50(A) and 50(B)) fromcompound (48) according to the invention (and includes the preparationof compound (47) from compound (41) for illustrative purposes).

[0125] According to a still further aspect of the invention there isprovided a process of synthetically producing a compound of the formula(1) and analogues and derivatives thereof which includes the steps ofsynthesising a suitable steroid intermediate or precursor and couplingthe required number of suitable monosaccharides with the steroidintermediate.

[0126] The invention also provides a process of coupling amonosaccharide cymarose with the steroid intermediate, which includesthe steps of

[0127] (i) reacting a cymarose moiety (38) with a steroid intermediate(15), e.g. at −15° C., and in the presence of tin chloride, in asolvent, e.g. ether, to produce a compound3-0-[4-0-benzoyl-2-phenylthio-β-D-cymaropyranosyl]-12,14-β-dihydroxy-pregn-5-ene-20-oneof the formula

[0128] and (ii) treating the compound (51) with tiglic acid chloride inpyridine and thereafter with a base, e.g. NaOMe, to produce a compound3-0-[-2-phenylthio-β-D-cymaropyranosyl]-12β-tigloyloxy14-hydroxy-14β-pregn-5-ene-20-one of the formula

[0129] The invention extends to a process which includes coupling amonosaccharide cymarose moiety to a monosaccharide thevetose moiety andcoupling the resultant disaccharide with the combined steroid product(52) to form compound (1).

[0130] The process of coupling the monosaccharide cymarose moiety to themonosaccharide thevetose moiety and coupling the resultant disaccharideto the combined steroid product (52) may include the steps of

[0131] (i) coupling a selectively protected cymarose moiety (40) and aselectively protected thevetose moiety (50 A) using tin chloride (SnCl₂)and silver trifluoromethanesulphonate, e.g. at −15° C., to produce acompound of the formula

[0132] in which Z=TBDMS=t-butyldimethylsilyl

[0133] (ii) treating compound (53) with tetrabutylammoniumfluoride toproduce a compound of the formula

[0134] (iii) treating compound (54) with diethylaminosulphurtrifluoride, e.g. at 0° C., to produce a compound of the formula

[0135] (iv) reacting compound (55) with compound (52) to produce acompound of the formula

[0136] and (v) treating compound (56) in a Raney-Nickel reaction andthereafter with a base, e.g. NaOMe, to produce compound (1) as describedabove.

[0137] Reaction Scheme E shows the procedure for the synthesis ofintermediates (52) and (55) and coupling them to form compound (56).

[0138] According to the invention, an alternative process is providedwhich includes coupling cymarose and thevetose moieties to form atrisaccharide and coupling the trisaccharide onto a steroid derivativeto form a compound of the formula (1).

[0139] The process of forming the trisaccharide and coupling theresultant trisaccharide to a steroid derivative may include the steps of

[0140] (i) coupling a selectively protected cymarose moiety (40) andcompound (45) using tin (II) chloride, AgOTf, Cp₂ZrCl₂ to produce acompound of the formula

[0141] in which Z=TBDMS=t-butyldimethylsilyl

[0142] (ii) treating compound (57) with tetrabutylammoniumfluoride anddiethylaminosulphur trifluoride to produce a trisaccharide compoundhaving the formula

[0143] and (iii) coupling the trisaccharide (58) with a steroidintermediate of the formula

[0144] using tin (II) chloride, AgOTf, Cp₂ZrCl₂ to produce compound (1).

[0145] The steroid intermediate (59) may be produced by treating steroid(15) with tiglic acid chloride.

[0146] Reaction Scheme F shows the procedure for the synthesis of thetrisaccharide (58) and the synthesis of compound (1) by coupling thetrisaccharide (58) with the steroid intermediate (59).

[0147] The intermediates (23), (24), (25), (27), (28), (29), (30), (37),(38), (39), (40), (48), (49), (50), (51), (53), (54), (55), (56), (57)and (58) described above are novel compounds and the invention extendsto these compounds as such.

[0148] Compound (1),3-0-[-β-D-thevetopyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl]-12β-0-tigloyloxy-14-hydroxy-14β-pregn-5-en-20-one,and various analogues and derivatives thereof have been found to haveappetite suppressing activity.

[0149] The invention extends also to a composition or formulation havingappetite suppressant activity, in which the active ingredient is anextract obtained from a plant of the genus Trichocaulon or the genusHoodia.

[0150] The active ingredient may be a compound of the formula (1),extracted from a plant of the genus Trichocaulon or Hoodia or aderivative thereof. The plant may be of the species Trichocaulonofficinale or Trichocaulon piliferum, or the species Hoodia currorii,Hoodia gordonii or Hoodia lugardii.

[0151] The invention extends also to a composition or formulation havingappetite suppressant activity, in which the active ingredient is asynthetically produced compound of the formula (1) or a derivative oranalogue thereof, as hereinbefore set out with reference to compounds(2) to (14).

[0152] According to another aspect of the invention there is provided amethod of suppressing an appetite by administering to a human or animala suitable dosage of an appetite suppressant agent comprising an extractof a plant of the genus Trichocaulon or Hoodia. The extract may beincorporated in a composition or formulation including alsopharmaceutically acceptable other ingredients.

[0153] The appetite suppressant agent may be an isolated naturalchemical or a synthetic chemical compound of the formula:

[0154] or derivatives or analogues thereof, as set out before.

[0155] The appetite suppressant composition or formulation may consistof the appetite suppressant agent admixed with a pharmaceuticalexcipient, diluent or carrier. Other suitable additives, including astabilizer and such other ingredients as may be desired may be added.

[0156] The invention extends to the use of compound (1) or itsderivatives or analogues in the manufacture of a medicament havingappetite suppressant activity.

[0157] The invention further extends to compound (1), or its derivativesor analogues as set out before, for use as a medicament having appetitesuppressant activity.

[0158] A method of suppressing an appetite by administering to a humanor animal an effective dosage of a composition as described above isalso provided.

[0159] A method has been described herein for extracting a steroidalglycoside having appetite suppressant activity from plant materialobtained from a plant of the Trichocaulon or Hoodia genus. The inventionthus extends to an extract obtained from plant material of theTrichocaulon or Hoodia genus and containing a substantially puresteroidal glycoside of formula (1).

[0160] The invention extends also to a foodstuff or a beveragecontaining an effective quantity of the steroidal glycoside of theformula (1), or its derivatives or analogues as set out before, to havean appetite suppressant effect when ingested.

[0161] Molecular genetic studies have led to a considerable increase inthe understanding of the regulation of appetite, satiety and bodyweight.These studies have revealed numerous central regulatory pathways,mediated by a number of neuropeptides. The maintenance of a normal bodyweight is achieved by an intricate balance between energy intake, foodconsumption, and energy expenditure. Energy homeostasis is subject to awide range of influences, ultimately controlled by the brain. Thedifferent signals include such things as sense of smell and taste andgastrointestinal signals such as distension of the gastrointestinaltract, chemical signals to the gastric mucosa and blood-bornemetabolites such as fatty acids and glucose.

[0162] Centrally, neuropeptide “Y” (NPY) which is negatively regulatedby leptin, has been established as one of the positive regulators offeeding behaviour. Expression of the endogenous antagonist formelanocortin receptors has also been shown to be the basis for obesityin a particular model (the ob/ob mouse). Indeed deficiency at the MC4melanocortin receptor completely replicates the obesity syndrome. Othermediators which have been shown to have roles in the energy balanceinclude bombesin, galonin and glucagon-like peptide-1.

[0163] Without being bound by theory, the Applicant believes thatcompound (1) and its analogues as described above act as an agonist ofthe melanocortin 4 receptor. The effect of this is to regulate NPY butalso to increase cholecystokinin. The effect of cholecystokinin amongstother things is to inhibit gastric emptying.

[0164] Accordingly, the invention extends to a composition havingappetite suppressant activity comprising a melanocortin 4 receptoragonist.

[0165] The agonist may be an extract or compound as previouslydescribed, in particular the compound of formula (1). The compositionmay be admixed with a pharmaceutical excipient, diluent or carrier andis optionally prepared in unit dosage form.

[0166] The invention still further extends to the use of a melanocortin4 receptor agonist in the manufacture of a medicament having appetitesuppressant activity, to a melanocortin 4 receptor agonist for use as amedicament having appetite suppressant activity, to a method ofsuppressing an appetite by administering to a human or animal aneffective dosage of a composition comprising a melanocortin 4 agonist asdescribed above, and to the use of a melanocortin 4 receptor agonist tosuppress the appetite of and/or to combat obesity in a human or animal.

[0167] The invention and its efficacy will now be further described,without limitation of the scope of the invention, with reference to thefollowing examples and drawings.

[0168] In the drawings,

[0169]FIG. 1 shows a flow diagram of the general method of extracting afirst crude appetite suppressant extract and a purified appetitesuppressant extract from plant material of the genus Trichocaulon orHoodia;

[0170]FIG. 2 shows a graphical representation of a bioassay carried outon rats using a partially purified methanol extract of Trichocaulonpiliferum;

[0171]FIGS. 3 and 4 together show a schematic representation of apreferred embodiment of the process of the invention for producing anextract of plant material of the genus Trichocaulon or Hoodia; and

[0172]FIGS. 5 and 6 show a graphical representation of the percentagechange of body mass of rats for different groups for days −7 to 7 anddays 0 to 7 respectively in a repeat dose study using a sap extract anda spray-dried sap extract of plant material of the species Hoodiagordonii.

EXAMPLE 1

[0173] The general method of extracting a first crude appetitesuppressant extract and a purified appetite suppressant extract fromplant material of the genus Trichocaulon or of the genus Hoodia isillustrated by way of the flow diagram of FIG. 1.

EXAMPLE 2

[0174] Bioassays carried out on rats using a partially purified methanolextract obtained in the manner illustrated in Example 1, indicated thatthe extract does in fact exhibit appetite suppressant activity. Theappetite suppressant activity of the active extract can be illustratedby way of a typical example of the effect of the methanol extract ofTrichocaulon piliferum on rats, by way of the graphic representation inFIG. 2.

[0175] It will be evident from FIG. 2 that the test group of rats dosedwith the extract on day 5 displayed a substantially diminished foodintake over the next two days, while a control group did not disclose acomparable reduced food intake. The food intake of the test groupreturned to normal, and in fact increased, from day 8 onwards.

EXAMPLE 3

[0176] A preferred embodiment of a process in accordance with theinvention for producing an extract having appetite suppressant activityis illustrated schematically by way of example in FIGS. 3 and 4, whichtwo Figures together illustrate the comprehensive process. However,various other procedures may be used, as will be understood by personsskilled in the art.

[0177] Referring to FIG. 3, plant material of the genus Trichocaulon orthe genus Hoodia is fed into a blender 3, eg a Waring blender, by way offeedline 1, with a solvent in the form of a methylene chloride/methanolsolution introduced via feedline 2. The homogenised product is fed vialine 4 into a separation stage 5, eg in the form of a filter orcentrifuge, and the residual plant material is removed via line 27.

[0178] The solvent/extract mixture is fed via line 6 into an evaporationstage 7, where the solvent is removed, for example by means of a rotorevaporator. The dried crude extract is fed via line 8 into a furtherextraction stage 9 with the addition of a methylene chloride/watersolution introduced via feedline 29 for further extraction, and then toa separation stage 13 by way of line 11, where the water fraction isremoved via line 31. The dissolved extract fraction is fed via line 15into a drier stage 17 where the solvent is evaporated, for example by arotor evaporator.

[0179] Referring to FIG. 4, the dried extract is fed via line 10 into anextraction stage 12. A methanol/hexane solution is also fed via line 14into the extraction stage 12 for further purification and extraction ofthe dried extract. The extract/methanol/hexane mixture is fed via line16 into a separation stage 18, the hexane fraction is removed via line20, and the methanol/extract mixture, is then fed via line 22 into adrying stage 24. In the drying stage 24, the solvent is removed, eg byevaporation on a rotor evaporator.

[0180] The dried, partially purified active extract is fed via line 26and with the addition of methanol via line 28 into a solution stage 30,and the dissolved fraction is fed via line 36 to a chromatography column38.

[0181] In the column 38 the methanol soluble fraction is furtherfractionated, using silica gel and a chloroform/30% methanol solvent,into different fractions schematically indicated as fractions I to V.According to an actual fractionation procedure carried out by theApplicant, the fractionation procedure yielded the following fractionweights: I(3.9 g); II(2.6 g); III(2.1 g); IV(1.1 g) and V(2.0 g). Thesefractions are individually evaluated by a suitable bioassaying procedure(in a step not shown) and those fractions identified as fractions I andII, displaying marked appetite suppressant activity, are fed byfeedlines 40 and 42 into columns 44 and 46 respectively where they arefurther fractionated and purified by column chromatography, again byusing silica gel and a 9:1 chloroform:methanol system.

[0182] The sub-fractions II(A)-(C) obtained from column 44 do not, whenassayed, display a noteworthy appetite suppressant activity, and may berecycled for further chromatography.

[0183] The sub-fractions I(A)-(L) obtained from column 46 are alsoevaluated (by an assaying step not shown), and the sub-fraction I(C) isfound to have marked appetite suppressant activity.

[0184] The sub-fraction I(C) is fed-via line 48 into column 50 for afurther fractionation and purification, using silica gel and a 9:1 ethylacetatehexane eluent. Of the resultant purified fractions, fractionI(C)(ii) is found, after assaying, to possess marked appetitesuppressant activity.

[0185] The purified product is identified by nuclear magnetic resonancespectroscopy (as indicated in Tables 1 and 2 below), to be compound (1).TABLE 1 ¹H (300.13 MHz) n.m.r. data for compound (1) CDCl3 Compound (1)Hydrogen Atom J(HH)/Hz δ_(H)/p.p.m. Aglycone-3 — 3.522 m 6 — 5.381 m 1211.5, 4.1 4.607 dd 17 9.3, 9.3 3.157 dd 18 — 1.029 s 19 — 0.951 s 21 —2.164 s 3* 7.1, 1.5 6.888 qq 4* 7.1, 1.2 1.806 dq 5* 1.6, 1.2 1.853 dqCym-1′ 9.4, 2.1 4.816 dd 2′_(aq) 13.8, 3.7, 2.1 2.055 ddd 2′_(ax) 13.8,9.4, 2.6 1.552 ddd 3′ 3.7, 2.9, 2.6 3.776 ddd 4′ 9.4, 2.9 3.179 dd 5′6.3, 9.4 3.821 dd 6′ 6.3 1.279 d^(a) 3′-OMe — 3.408 s^(d) 1″ 9.4, 2.14.730 dd 2″ 13.8, 3.7, 2.1 2.108 ddd 2″^(aq) 13.8, 9.4, 2.6 1.601 ddd3″^(ax) 3.7, 2.9, 2.6 3.755 ddd 4″ 9.4, 2.9 3.239 dd 5″ 6.3, 9.4 3.898dd 6″ 6.3 1.243 d^(b) 3″-OMe — 3.392 s^(e) Thev-1″′ 7.7 4.273 d 2″′ 7.7,8.0 3.469 dd 3″′ 8.0, 2.9 3.099 dd 4″′ 9.3, 2.9 3.179 dd 5″′ 6.3, 9.33.351 dd 6″′ 6.3 1.183 d′^(c) 3″′-OMe — 3.622 s

[0186] TABLE 2 Relevant ¹³C (75.25 MHz) n.m.r. data for Compound (1) inCDCl3 Aglycone moiety Sugar moiety Carbon δ_(c)/p.p.m. Carbonδ_(c)/p.p.m. 1 37.04 T cym- 1′ 95.84 D 2 29.44 T 2′ 35.57 T 3 77.24 D 3′77.05 D 4 38.62 T 4′ 82.57 D 5 138.95 S 5′ 68.48 D 6 131.90 D 6′ 18.14 Q7 27.30 T 3′-OMe 57.93 Q 8 35.30 D 1″ 99.54 D 9 43.04 D 2″ 35.17 T 10 37.22 S 3″ 76.99 D 11  26.04 T 4″ 82.52 D 12  75.88 D 5″ 68.30 D 13 53.71 S 6″ 18.36 Q 14  85.69 S 3″-OMe 57.09 Q 15  34.36 T Thev- 1″′104.28 D 16  24.31 T 2″′ 74.62 D 17  57.18 D 3″′ 85.30 D 18  9.85 Q 4″′74.62 D 19  19.27 Q 5″′ 71.62 D 20  216.85 S 6″′ 17.75 Q 21  33.01 Q3″′-OMe 60.60 Q  1* 167.60 S  2* 128.69 D  3* 137.66 D  4* 14.41 Q  5*12.08 Q

[0187] Compound (1)

[0188] IR data: 3440 cm⁻¹ (OH), 2910 cm⁻¹ (CH), 1700 cm⁻¹ (C=0) [α_(D])²⁰ ₅₈₉=12,67° (C=3,CHCl₃) m.p. 147° C.-152° C.

[0189] Examples 4 to 13 illustrate the synthetic procedures whereby theintermediate compounds and steroid (15) may be prepared according to“the first alternative procedure”.

EXAMPLE 4 12β15α-Dihydroxy progesterone (17)

[0190] Cultures of Calonectria decora (ATCC 14767) are prepared by theinoculation of a culture medium comprised of sucrose (900 g), K₂HPO₄ (30g), Czapek concentrate (300 ml), corn steep liquor (300 ml) anddistilled water (30 l) (150×500 ml flasks). After 5 days of shaking at26° C., progesterone (16) (150 g) in a suspension of Tween 80 (0,1%soln., 1,5 l) is added to the flasks. The cultures are incubated for afurther 5 days and then worked-up by centrifugation, decantation,extraction of the medium with chloroform, and then evaporation to yieldthe dihydroxy. progesterone (17) (75 g, 45%)

[0191]¹H NMR (CDCl₃): 5, 71 (1H, s, H-4); 4,12-4,22 (1H, m, H-15) 4,43(1H, br, s, OH); 3,46-3,53 (1H, dd, J=4,6Hz, H-12); 2,16 Hz (3H, s,H-21); 1,18 (3H, s, H-19); 0,74 (3H, s, H-18)

EXAMPLE 5 12β-Hydroxy-15α-(p-toluene sulfonyl)-progesterone (18)

[0192] The dihydroxy progesterone (17) (75 g, 0.22 mol) is dissolved indry pyridine (300 ml) and cooled to 0° C. p-Toluene sulfonyl chloride(46 g, 0,24 mol) in dry pyridine (200 ml) is added dropwise to thereaction mixture at 0° C. The reaction is stirred overnight at 0° C.,and quenched by the addition of H₂O (500 ml). The water layer isextracted with ethyl acetate (1 l), and the organic extract washed withhydrochloric acid (6M, 3×1 l), aqueous saturated sodium bicarbonate (500ml), aqueous saturated sodium chloride (500 ml), and water (500 ml). Theorganic layer is dried (MgSO₄), filtered and evaporated to yieldp-toluene sulfonated progesterone (18) (98 g, 92%) as a viscous darkyellow oil.

[0193]¹H NMR (CDCl₃): 7,7 (2H, d, J=14 Hz, H-2,6); 7,34 (2H, d, J=8,4Hz, H-3,5); 5,67 (1H, s, H-4); 4,86-4,93 (1H, m, H-15); 3,45-3,50 (1H,dd, J=4,6 Hz, H-12); 2,44 (3H, s, H-4Me); 2,15 (3H, s, H-21) 1,13 (3H,s, H-19); 0,74 (3H, s, H-18).

EXAMPLE 6 12β-Hydroxy-Δ¹⁴-progesterone (19)

[0194] A solution of the tosylated progesterone (18) (98 g, 0,19 mol) in2,4,6-trimethyl collidine (500 ml) is refluxed at 150° C. for 3 h. Thereaction mixture is cooled and poured into water (500 ml). The waterlayer is extracted with ethyl acetate (1 l), after which the organiclayer is washed with hydrochloric acid (6M, 3×1 l), aqueous saturatedsodium bicarbonate (500 ml), aqueous saturated sodium chloride (500 ml),and water (500 ml). After drying (MgSO₄) and filtering, the ethylacetate is evaporated and the crude mixture is purified by silica gelchromatography, eluting with acetone: chloroform (1:10) to affordΔ¹⁴-progesterone (19) (50 g, 78%) as a dark red oil.

[0195]¹H NMR (CDCl₃): 5,73 (1H, s, H-4), 5,28 (1H, dd, J=2,2 Hz, H-15),4,41 (1H, br, s, OH), 3,49-3,52 (1H, dd, J=4,3 Hz, H-12), 2,80-2,84 (1H,dd, J=9,2 Hz, H-17), 2,14 (3H, s, H-21), 1,19 (3H, s, H-19), 0.89 (3H,S, H-18).

EXAMPLE 7 3,12β-Diacetoxypregna-3,5,14-trien-20-one (20)

[0196] A solution of Δ¹⁴-progesterone (19) (50 g, 0,15 mol) in acetylchloride (1,5 l) and acetic anhydride (750 ml) is refluxed for 2 hours.The reaction mixture is poured into cold ethyl acetate (1 l) and aqueoussaturated sodium bicarbonate is added with stirring until theeffervescence ceases. The ethyl acetate layer is separated from thesodium bicarbonate layer and washed with further portions of aqueoussodium bicarbonate (3×700 ml), thereafter with aqueous saturated sodiumchloride (700 ml) and finally with water (700 ml). The organic layer isdried (MgSO₄), filtered and evaporated to afford the3,12β-diacetoxypregna-3,5,14-trien-20-one-(20) (60 g, 93%) as an orangeoil.

[0197]¹H NMR(CDCl₃): 5,68 (1H, s, H-4), 5,44 (1H, m, H-6), 5,31 (1H, dd,J=2,2 Hz, H-15), 4,82-4,86 (1H, dd, J=4,5 Hz, H-12), 3,10-3,18 (1H, t,J=9,5 Hz, H-17), 2,18 (3H, s, 3-Ac), 2,11 (3H, s, 12-Ac), 2,08 (3H, s,H-21), 1,02 (3H, s, H-19), 1,01 (3H, s, H-18)

EXAMPLE 8 3,12β-Diacetoxy-20,20-ethylenedioxypregna-3,5,14-triene (21)

[0198] The diacetoxy compound (20) (60 g, 0,14 mol) is dissolved inbenzene (1 l) and ethylene glycol (60 ml) and p-toluene sulfonic acid (1g) are added. (The benzene is previously refluxed with a Dean-Starktrap). The mixture is refluxed with stirring and azeotropic removal ofwater for 16 hours. Aqueous saturated sodium bicarbonate solution (500ml) is added to the cooled solution. This is then washed with brine (500ml), and with water (500 ml), and dried (MgSO₄). The solvent isevaporated and the crude mixture purified by silica gel columnchromatography, eluting with ethyl acetate: hexane (2:8) to yield theethylenedioxypregna-3,5,14-triene (21) (35 g, 53%).

[0199]¹H NMR (CDCl₃): 5,68 (1H, s, H-4), 5,45 (1H, m, H-6), 5,31 (1H,dd, J=2,2 Hz, H-15), 4,73-4,85 (1H, dd, J=4,4 Hz, H-12), 3,78-3,98 (4H,m, ethylenedioxy), 2,16 (3H, s, 3-Ac), 2,04 (3H, s, 12-Ac), 1,29 (3H, s,H-21), 1,12 (3H, s, H-19), 1,02 (3H, s, H-18).

EXAMPLE 9 3β-12β-Dihydroxy-20,20-ethylenedioxypregna-5,14-diene-12acetate (22)

[0200] The dienolacetate (21) (35 g, 0,077 mol) is suspended in ethanol(500 ml) and sodium borohydride (2,8 g, 0.074 mol) is added at 0° C. Themixture is allowed to warm to room temperature and stirred overnight.Most of the solvent is removed in vacuo and the mixture is diluted withwater (500 ml) and extracted with ethyl acetate (500 ml). Work-upfollowed by chromatography on silica gel with acetone/chloroform (1:10)yields the 3β-alcohol (22) (25 g, 80%).

[0201]¹H NMR (CDCl₃): 5,41 (1H, m, H-6), 5,28 (1H, dd, J=2,2 Hz, H-15),4,72-4,81 (1H, dd, J=4,4 Hz, H-12), 3,82-4,02 (4H, m, ethylene dioxy),3,45-3,59 (1H, m, H-3), 2,03 (3H, s, 12-Ac), 1,28 (3H, s, H-21), 1,10(3H, s, H-19), 1,01 (3H, s, H-18).

EXAMPLE 10 3β,12β-Dihydroxy-20,20-ethylenedioxypregn-5,14-diene (23)

[0202] The 3β-alcohol (22) (25 g, 60.2 mmol) in dry tetrahydrofuran (300ml) is added dropwise to a suspension of lithium aluminium hydride (2,7g, 72,2 mmol) in dry tetrahydrofuran (500 ml). The reaction mixture isstirred at room temperature for 24 hours after which water (2,7 ml) iscarefully added and stirred for a further 10 min. Sodium hydroxide (15%soln, 2,7 ml) is then added and the suspension stirred. After 10 min,water (8,1 ml) is added and the suspension stirred for 10 minutes,filtered, dried (MgSO₄), and the solvent evaporated to afford the 3β,12βdihydroxypregna-diene (23) (20 g, 90%).

[0203]¹H NMR (CDCl₃): 5,36 (1H, m, H-6), 5,23 (1H, dd, J=2,2 Hz, H-15),3,94-4,06 (4H, m, ethylene dioxy), 3,41-3,52 (1H, m, H-3), 3,32-3,36(1H, dd, J=4,3 Hz, H-12), 1,31 (3H, s, H) 1,01 (3H, s, H-19), 0,96 (3H,s, H-18).

[0204]¹³C NMR (CDCl₃): 152,4 (c-14), 140,2 (c-5), 121,1 (c-15) 119,7(c-6), 111,1 (C-20), 79,8 (C-12), 71,6 (C-3), 63,7 and 63,6 (ethylenedioxy), 58,8 (C-17), 19,0 (C-19), 11,9 (C-18).

3β,12β-Dihydroxy-14,15-epoxy-20,20-ethylenedioxypregn-5-ene;3β,12β-Dihydroxy-5,6-epoxy-20,20-ethylenedioxypregn-14-ene

[0205] N-Bromoacetamide (211 mg, 1,5 mmol) is added to a stirredsolution of the 5,14-diene (23) (500 mg, 1,34 mmol) in acetone (100 ml),acetic acid (2,5 ml), and water (5 ml) at 0° C. After 15 min sodiumsulphite (5% soln, 50 ml) is added to the reaction mixture. The acetoneis evaporated, and the aqueous layer extracted with dichloromethane(3×50 ml). The organic layer is dried (MgSO₄), filtered and evaporated.Pyridine (1 ml) is added to the product, and stirred for 0,5 h.Dichloromethane (100 ml) is then added to the reaction mixture, and thedichloromethane is washed with citric acid (5% soln, 3×100 ml),saturated sodium bicarbonate (50 ml), and water (50 ml). The organiclayer is dried (MgSO₄), filtered and evaporated to give the mixture of14,15- and 5,6-epoxides (360 mg, 69%) as a white foam. The mixture ofepoxides could not be separated by silica gel column chromatography.

EXAMPLE 11 3β,12β-Dihydroxy-14,15-epoxy-20,20-ethylenedioxypregn-5-ene(24)

[0206] The mixture of 14,15- and 5,6-epoxides (14,4 g, 37,0 mmol) in drytetrahydrofuran (200 ml) is added to a suspension of lithium aluminiumhydride (1,69 g, 44,4 mmol) in dry tetrahydrofuran (300 ml). Thereaction mixture is stirred at room temperature for 24 hours, afterwhich it is worked up as described earlier by the addition of water(1,69 ml), and sodium hydroxide (15% soln, 1,69 ml). After filtrationand evaporation of the solvent, the crude product is purified by silicagel column chromatography using methanol/chloroform (1:9) as solvent togive the unreacted 14,15 epoxy-20,20-ethylenedioxypregn-5-ene (24) (300mg, 2,1%).

[0207]¹H NMR (CDCl₃): 5,31 (1H, m, H-6), 3,82-3,98 (4H, m, ethylenedioxy), 3,43-3,52 (1H, m, H-3), 3,41 (1H, s, H-15), 3,31-3,35 (1H, dd,J=4,3 Hz, H-12), 1,29 (3H, s, H-21), 1,17 (3H, s, H-19), 1,02 (3H, s,H-18)

[0208]¹³C NMR (CDCl₃): 139,8 (C-5), 120,8 (C-6), 112,1 (C-20), 77,2(C-12), 75,4 (C-14), 61,0 (C-15), 22,3 (C-21), 19,2 (C-19), 9,5 (C-18).

EXAMPLE 12 3β,12β,14β-Trihydroxy-20,20-ethylenedioxypregn-5-ene (25)

[0209] The 14,15-epoxide (24) (300 mg, 0,77 mmol) in dry tetrahydrofuran(10 ml) is added to a suspension of lithium aluminium hydride (300 mg,7,89 mmol) in tetrahydrofuran and the reaction refluxed for 48 h. Afterthe addition of water (0,3 ml), sodium hydroxide (15% soln, 0,3 ml) andfiltration as described earlier, the mixture is purified by silica gelcolumn chromatography using methanol:chloroform (1:9) as solvent to givethe trihydroxy pregnene (25) (250 mg, 83%).

[0210]¹H NMR (CDCl₃):5,38 (1H, m, H-6), 3,98 (4H, m, ethylene dioxy),3,43-3,53 (1H, m, H-3), 3,25-3,32 (1H, dd, J=4,1 Hz, H-12), 1,32 (3H, s,H-21), 1,01 (3H, s, H-19), 0,98 (3H, s, H-18)

[0211]¹³C NMR CDCl₃): 139,1 (C-5), 122,1 (C-6), 112,2 (C-20), 85,1(C-14), 75,1 (C-12), 71,6 (C-3), 23,4 (C-21), 19,4 (C-19), 8,9 (C-18)

EXAMPLE 13 3β,12β,14β-Trihydroxy-pregn-5-ene (15)

[0212] The ethylenedioxypregnene (25) (250 mg, 0,64 mmol) is dissolvedin acetic acid (13,4 ml) and water which after freeze drying affords thetrihydroxy steroid (15) (200 mg, 89%), m.p.: 228°-235° C. (lit.225°-235° C.), M+348, [α_(D)]²⁰ +35° (lit [α_(D)]²⁰+29°).

[0213]¹H NMR (CDCl₃): 5,39 (1H, m, H-6), 3,56-3,62 (1H, t, J=8,1 Hz,H-17), 3,42-3,51 (1H, m, H-3), 3,28-3,39 (1H, dd, J=4,3 Hz, H-12), 2,23(3H, s, H-21), 1,01 (3H, s, H-19), 0,90 (3H, s, H-18)

[0214]¹³C NMR (CDCl₃): 217,7 (C-20), 138,9 (C-5), 122,2 (C-6), 85,5(C-14), 73,6 (C-12), 71,6 (C-3), 57,0 (C-17), 55,1 (C-13), 43,6 (C-9),42,1 (C-4), 37,3 (C-1), 36,8 (C-10), 35,9 (C-8), 34,5 (C-15), 32,9(C-21), 31,5 (C-16), 30,1 (C-2), 27,4 (C-7), 24,4 (C-11), 19,4 (C-19),8,3 (C-18).

[0215] Examples 14 to 19 illustrate the synthetic procedures whereby theintermediate compounds and steroid (15) may be prepared according to“the second alternative procedure”.

EXAMPLE 14

[0216]20,20-Ethylenedioxy-3β-toluene-p-sulphonyloxy-pregn-5,14-diene-12β-olacetate (26)—A solution of p-toluenesulphonyl chloride (650 mg, 3.4mmol) in pyridine (10 ml) was added dropwise to a mixture of the20,20-Ethylenedioxypregna-5,14-diene-3β,12β-diol 12-acetate (22) (1.3 g,3.1 mmol) in pyridine (15 ml) at 0° C. The reaction mixture was leftstirring at room temperature for 24 hours after which water was added tothe reaction mixture. The solution was extracted with ethyl acetate(2×50 ml), the ethyl acetate layer was washed citric acid (5×50 ml),saturated sodium bicarbonate solution (100 ml), saturated sodiumchloride solution (100 ml) and water (100 ml). The ethyl acetate wasdried (MgSO₄), filtered, and evaporated and purified by flash columnchromatography using hexane-ethyl acetate (8:2 v/v) as the eluant togive the β-O-tosyl steroid (26), (1.5 g, 84%), as a yellow oil, (Found M570.271, C₃₂H₄₂O₇S requires: M 570.273).

[0217] δ_(H) 1.021 (3H, s, 19-H), 1.131 (3H, s, 18-H), 1.282 (3H, s,21-H), 2.021 (acetate0CH₃), 2.431 (3H, s, Ar-CH₃), 3.883 (4H, m,OCH₂CH₂O), 4.750 (1H, dd, ³ J 10.8 Hz, 5.2 Hz, 12-H), 4.890 (1H, m,30H), 5.281 (1H, dd, ³ J 4.2 Hz, 2.1 Hz, 15-H), 5.388 (1H, m, 6-H),7.341 (2H, d,³ J 8.2 Hz, ArH), 7.746 (2H, d, ³ J 8.2 Hz, ArH).

[0218] δ_(c) 13.493Q (C-18), 19.002Q (C-19), 21.612Q (Ar-methyl)*,21.671Q (C-21)*, 24.175Q (acetate methyl), 63.401T (ethylenedioxy),63.498T (ethylenedioxy), 71.531S (C-13), 80.912D (C-12), 82.531D (C-3),111.363S (C-20), 120.881D (C-15), 121.461D (C-6), 123.715-133.917(Aromatic), 139,903S (C-14), 151,722S (C-5) 170.819S (ester carbonyl). *may be interchanged

EXAMPLE 15

[0219]20,20-Ethylenedioxy-3α,5-cyclo-5α-pregn-14-ene-6β,12β-diol-12-acetate(27)—A solution of 3β-toluene-p-sulphonyloxy-pregn-5, 14-diene (26) (1.2g, 2.1 mmol) and potassium acetate (2.2 g, 22.4 mmol) in water (250 ml)and acetone (500 ml) was refluxed at 60° C. for 16 hours. The acetonewas evaporated and the water was extracted with ethyl acetate (200 ml).The ethyl acetate was dried (MgSO₄), filtered, and evaporated. Flashchromatographic separation of the mixture using chloroform-acetone (9:1v/v) as the eluant gave the 3α,5-cyclo derivative (27), (530 mg, 610%)as a yellow oil, (Found M 416.262, C₂₅H₃₆O₅ requires: M 416.263).

[0220] δ_(H) 0.288 (1H, dd, ³ J 8.1 Hz, 4.9 Hz, 4-H_(a)) 0.477 (1H, dd,³ J 4.4 Hz, 4.4 Hz, 4-H_(b)), 1.025 (3H, S, 19-H), 1.121 (3H, s, 18-H),1.256 (3H, s, 21-H), 1.989 (3H, s, acetate-CH₃), 3.302 (1H, dd, ₃ J 2.8Hz 2.8 Hz, 6-H), 3.784-3.947 (4H, m, OCH₂CH₂O), 4.721 (1H, dd, ³ J 8.5Hz, 5.6 Hz, 12-H), 5.232 (1H, dd, ³ J 3.9 Hz, 1.9 Hz, 15-H).

[0221] δ_(c) 11.678T(C-4), 12.298Q(C-18), 19.971Q (C-19), 23.623Q(C-21),24.153Q (acetate methyl), 63.700T (ethylenedioxy), 63.788T(ethylenedioxy), 73.591D (C-6), 80.551D (C-12), 111.126S (C-20),118.778D (C-15), 152.959S (C-14), 170.991S (ester carbonyl).

EXAMPLE 16

[0222] 20,20-Ethylenedioxy-3α,5-cyclo-5α-pregn-14-ene-6β,12β-diol (28)—Asolution of the 3α, 5-cyclo derivative (27), (500 mg, 1.2 mmol) intetrahydrofuran (20 ml) was added dropwise to a suspension of lithiumaluminium hydride (50 mg, 1.3 mmol) in tetrahydrofuran (10 ml). Thereaction mixture was stirred for 4 hours and quenched by the addition ofwater (50 μl). After 30 minutes, sodium hydroxide was added (15%solution, 50 μl) and stirring continued for a further 30 minutes. Water(150 μl was added and the reaction mixture was filtered. Thetetrahydrofuran was dried (MgSO₄) filtered and evaporated and flashchromatographic purification using chloroform-acetone (8:2 v/v) as theeluant to give the diol (28), (370 mg, 83%) as an oil, (Found M 374.250,C₂₃H₃₄O₄ requires: M 374.252)

[0223] δ_(H) 0.298 (1H, dd, ³ J 8.1 Hz, 4.9 Hz, 4-H₂), 0.510 (1H, dd, ³J 4.4 Hz, 4.4 Hz, 4-H_(b)), 0.985 (3H, s, 19-H), 1.055 (3H, s, 18-H),1.325 (3H, s, 21-H), 3.318 (1H, dd, ³ J 3.0 Hz, 3.0 Hz, 6-H),), 3.363(1H, dd, ³ J 11.4 Hz, 4.2 Hz, 12-H), 4.019 (4H, m, OCH₂Ch₂O) 4.622 (1H,s, OH), 5.255 (1H, dd, ³ J 3.9 Hz, 1.9 Hz, 15-H)

[0224] δ_(c) 11.681T(C-4), 12.243Q(C-18), 19.844Q (C-19), 23.604Q(C-21),63.620T (ethylenedioxy), 63.733T (ethylenedioxy), 73.569D (C-6), 77.478D(C-12), 111.125S (C-20), 118.702D (C-15), 152.912S (C-14).

EXAMPLE 17

[0225] 20,20-Ethylenedioxy-14,15β-epoxy-3α,5-cyclo-5α,14β-pregnane-6δ,12β-diol (29) -N-bromoacetamide (150 mg, 1.1mmol) was added to a solution of the20,20-ethylenedioxy-3α,5-cyclo-5α-pregn-14-ene-6β, 12β-diol (28) (340mg, 0.91 mmol) in acetone (20 ml), water (0.25 ml) and acetic acid (0.25ml) at 0° C. After 15 min., sodium sulphite (5% solution, 20 ml) wasadded to the reaction mixture. The acetone was evaporated under reducedpressure and the remaining solution was extracted with dichloromethane(3×30 ml). The dichloromethane layer was dried (MgSO₄), filtered andevaporated to a concentrated volume (50 ml). Pyridine (0.5 ml) was addedto the mixture and stirred for a further 1 hour after which thedichloromethane layer was washed with a citric acid solution (5%, 3×30ml), saturated sodium bicarbonate solution (30 ml) and water (30 ml).The dichloromethane layer was dried (MgSO)₄), filtered and evaporatedand purified by flash column chromatography using chloroform-methanol(9.5:0.5 v/v) as the eluant to give the epoxide (29) (180 mg, 51% as afoam, (Found M 390.245, C₂₃H₃₄O₂ requires: M 390.247).

[0226] δ_(H) 0.287 (1H, dd, ³ J 8.1 Hz, 4.9 Hz, 4-H_(a)), 0.501 (1H, dd,³ J 4.4 Hz, 4.4 Hz,4-H_(b)), 0.978 (3H, s, 19-H), 1.048 (3H, s, 18-H),1.321 (3H, s, 21-H), 3.318 (1H, dd, ₃ J 3.1 Hz, 3.1 Hz, 6-H), ), 3.355(1H, dd, 3 J 11.2 Hz, 4.1 Hz, 12-H), 3.491 (1H, s, 15-H), 4.001 (4H, m,OCH₂Ch₂O), 4.901 (1H, s, OH).

[0227] δ_(c) 11.668T(C-4), 11.973Q(C-18), 19.515Q (C-19), 23.519Q(C-21),59.910D (C-15), 63.601T (ethylenedioxy), 63.713T (ethylenedioxy),72.501S (C-14), 73.571D (C-6), 77.471D (C-12), 111.085S (C-20).

EXAMPLE 18

[0228] 20,20-Ethylenedioxy-6β, 12β, 14-trihydroxy-3α, 5-cyclo-5α,14β-pregnane (30)—A solution of the epoxide (29) (170 mg, 0.44 mmol) intetrahydrofuran (10 ml) was added to a suspension of lithium aluminiumhydride (20 mg, 0.53 mmol) in tetrahydrofuran (5 ml). The reactionmixture was refluxed for 2 hours after which water (20 μl) was added andstirring continued for 05 hour. Sodium hydroxide solution (15%, 20 μl)was added and stirring continued for a further 0.5 hour. A furtherquantity of water was added (60 μl) and the suspension was stirred for 1hour. After filtration, the suspension was dried (MgSO₄) filtered, andthe tetrahydrofuran was evaporated. Flash chromatographic separation ofthe resulting mixture eluting with chloroform-methanol (9:1 v/v) gavethe required triol (30), (90 mg, 53%) as a clear oil, (Found M 392.261,C₂₃H₃₈O₅ requires: M 392.263).

[0229] δ_(H) 0.287 (1H, dd, ³ J 8.1 Hz, 4.9 Hz, 4-H₂), 0.510 (1H, dd, ³J 4.4 Hz, 4.4 Hz, 4-H_(b)), 0.971 (3H, s, 19-H), 1.042 (3H, s, 18-H),1.319 (3H, s, 21-H), 3.321 (1H, dd, ³ J 3.0 Hz, 3.0 Hz, 6-H), 3.321 (1H,dd, ³ J 11.1 Hz, 3.9 Hz, 12-H), 3.561 (1H, s, OH), 4.084 (4h, m,OCH₂Ch₂O) 4.671 (1H, s, OH)

[0230] δ_(c) 11.668T(C-4), 11.971Q(C-18), 19.511Q (C-19), 23.520Q(C-21), 63.612T (ethylenedioxy), 63.711T (ethylenedioxy), 73.483D (C-6),76.051D (C-12), 84.307S (C-14), 111.099S (C-20).

EXAMPLE 19

[0231] 3β, 12β, 14-Trihydroxy-14β-pregn-5-en-20-one (15)—A mixture ofthe triol (30) (80 mg, 0.20 mmol) in acetone (20 ml) and hydrochloricacid (1M, 10 ml) was refluxed at 60° C. for 2 hours. The reactionmixture was cooled and saturated sodium bicarbonate solution *(20 ml)was added. The acetone was evaporated and the aqueous layer extractedwith chloroform (3×20 ml), the chloroform layer was dried (MgSO₄),filtered and evaporated to give the epimeric trihydroxy steroids (15a,15b) (42 mg, 61%). Separation of the epimeric mixture (15a, 15b) (15 mg)was achieved by flash chromatographic separation using chloroformmethanol (9:1 v/v) as the eluant to give the pure 17β-epimer (15a), (10mg), m.p. 224-229° C. (acetone), (lit. 226-223°), (Found M 348.234,C;72.32, H 9.21% C₂₁H₃₂O₄ requires: C, 72.38; H 9.26%, M 348.236), and the17α-epimer (15B) (3 mg), m.p. 183-191° C. (acetone), (lit 184-196°).

[0232] 3β, 12β, 14-Trihydroxy-14β-pregn-5-en-20-one (15a): δ_(H) 0.963(1H, s, 19-H), 1.192 (3H, s, 18-H), 2.236 (3H, s 21-H), 3.325 (1H, dd, ³J 11.2 Hz, 3.9 Hz, 12-H), 3.464 (1H, s, OH), 3.5140 (1H, m, 3-H), 3.598(1H, dd, ³ J 9.6 Hz, 9.6 Hz, 17-H), 4.255 (1H, s, OH), 5.383 (1H, m,5-H)

[0233] δ_(c) 8.275Q (C-18), 19.414Q (C-19), 24.400T (C-11) 24.581T(C-16), 27.443T (C-7), 30.062T (C-2), 32.972Q (C-21), 34.543T (C-15),35.864D (C-8), 36.975S (C-10), 37.337T (C-1), 42.144T (C-4), 43.565D(C-9), 55.101S (C-13), 57.038D (C-17), 71.597D (C-3), 73.558D (C-12),85.566S (C-14), 122.223D (C-6), 138.932S (C-5), 217.011S (C-20).

[0234] 3β, 12β, 14-Trihydroxy-14β-pregn-5-en-20-one (15b): δ_(H) 0.996(1H, s, 19-H), 1.144 (3H, s, 18-H), 2.221 (3H, s 21-H), 3.339 (1H, dd, ³J 9.4 Hz, 9.4 Hz, 17-H), 3.492 (1H, m, 3-H), 3.629 (1H, dd, ³ J11.1 Hz,3.9 Hz, 12-H), 3.712 (1H, s, OH), 4.325 (1H, s, OH), 5.383 (1H, m, 5-H).

[0235] Examples 20 to 28 illustrate the procedures whereby theintermediate compounds may be prepared to form the first. monosaccharide(40).

EXAMPLE 20 Methyl-4, 6-0-benzylidene-α-D-glucopyranoside (32)

[0236] A mixture of methyl-α-D-glucopyranoside (30 g, 0,15 mol),benzaldehyde (70 ml) and zinc chloride (20 g) is stirred at roomtemperature for 24 hours. The reaction product is poured into ice waterand stirring continued for 15 min. The white precipitate is filtered andwashed with diethyl ether. The solid material is stirred with a solutionof sodium metabisulphite (10% soln), for 15 min, filtered and washedwith water. The solid material is crystallized from chloroform and etherto yield the benzylidene product (32) (31 g, 72%).

EXAMPLE 21 Methyl-4,6-0-benzylidene-2-0-tosyl-α-D-glucopyranoside(33)

[0237] p-Toluene sulfonyl chloride (25 g, 1,2 eq) in pyridine (100 ml)is added dropwise to a solution of the benzylidene glucose (32) (31 g,0.12 mol) in pyridine (100 ml) at 0° C. The reaction is stirred at roomtemperature for 48 hours. Ice is added to the reaction mixture. Theresulting white solid material is washed with water and recrystallizedfrom hot ethanol to yield the tosylated glucose (33) (28 g, 60%).

EXAMPLE 22 Methyl-4,6-0-benzylidene-3-0-methyl-α-D-altropyranoside (34)

[0238] The tosylate (33) (28 g, 64 mmol) in a solution of is sodium (7g) in methanol (150 ml) is heated at 110° C. for 48 hour in anautoclave. The reaction vessel is cooled and solid carbon dioxide isadded to the reaction mixture. After filtration, the methanol isevaporated and the solid material is then taken up in water. The aqueouslayer is extracted with chloroform (×3). The chloroform is dried(MgSO₄), filtered and evaporated. The crude mixture is purified bysilica gel column chromatography eluting with chloroform : acetone (9:1)to yield the altroside (34) (10 g, 52% )

EXAMPLE 23Methyl-6-bromo-4-0-benzoyl-3-0-methyl-6-deoxy-α-D-altropyranoside (35)

[0239] The benzylidene altroside (34) (10 g, 33 mmol) is added to asolution of N-bromosuccinimide (7.6 g) and barium carbonate (20 g) incarbon tetrachloride and the reaction mixture is refluxed at 75° C. for3 hours. The reaction mixture is filtered and the carbon tetrachloridelayer is washed with water. The organic layer is dried (MgSO4), filteredand evaporated to yield 6-bromo-altroside (35), (9 g, 69%).

EXAMPLE 24 Methyl-4-0-benzoyl-3-0-methyl-6-deoxy-α-D-altropyranoside(36)

[0240] Sodium borohydride (18 g) in water (30 ml) is added dropwise to asolution of the bromoaltroside (35) (9 g, 23 mmol) and nickel chloride(18 g) in ethanol (300 ml) at 0° C. The reaction mixture is refluxed at75° C. for 1 hour and then it is filtered. The ethanol is evaporated andthe remaining aqueous layer is extracted with chloroform (×3). Thechloroform is dried (MgSO₄), filtered and evaporated, to yield the6-deoxy-altroside (3.6) (5 g, 72%).

EXAMPLE 25 4-0-Benzoyl-3-0-methyl-6-deoxy-αβ-D-phenylthioaltropyranoside(37)

[0241] Phenylthiotrimethylsilane (5 ml) andtrimethylsilyltrifluoromethane sulphonate (2 ml) are added at 0° C. to asolution of the 6-deoxy-altroside (36) (5 g, 17 mmol) in dichloromethane(200 ml). The reaction mixture is stirred at room temperature for 6hours. Saturated sodium bicarbonate is added to the reaction mixture.The dichloromethane layer is dried (MgSO₄), filtered and evaporated. Thecrude mixture is purified by silica gel column chromatography elutingwith chloroform:acetone (9:1) to yield the αβ-phenylthioaltroside (37)(4 g, 63%).

EXAMPLE 264-0-Benzoyl-3-0-methyl-2-phenylthio-2,6-dideoxy-αβ-D-fluorocymaropyranoside(38)

[0242] Diethylaminosulphurtrifluoride (0,65 g) is added rapidly to asolution of the αβ-phenylthioaltroside (37) (0,5 g, 1,33 mmol) indichloromethane at 0° C. The reaction is stirred for 0,5 h at 0° C. andthen saturated sodium bicarbonate is added. The dichloromethane isseparated from the aqueous layer, dried (MgSO₄), filtered and evaporatedto yield the αβ-fluorocymarose (38) (450 mg, 90%).

EXAMPLE 274-0-Benzoyl-3-0-methyl-2-0-t-butyldimethylsilyl-αβ-D-phenylthio-altroside(39)

[0243] The 6-deoxy altroside (37) (5 g) is silylated usingt-butyldimethylsilylchloride (3 g) and imidazole (3 g) in pyridine (50ml). The reaction is worked-up by extracting with ethyl acetate, washingthe ethyl acetate with hydrochloric acid (6 N), then with sodiumbicarbonate, and finally with water. The ethyl acetate layer is dried(MgSO₄), filtered and evaporated to yield the silylated benzoylphenylthioaltroside (39) (80%).

EXAMPLE 28 3-0-methyl-2-0-t-butyldimethylsilyl-αβ-D-phenylthioaltroside(40)

[0244] The silylated benzoyl phenylthioaltroside (39) (6 g) is treatedwith sodium methoxide (100 ml) for 4 hours. The methanol is evaporatedand water is added to the reaction. The water layer is acidified (pH 5,ACOH) and extracted with ethyl acetate. The ethyl acetate is washed withwater, dried (MgSO₄), filtered and evaporated to yield silylated methylphenylthioaltroside (40) (75%).

[0245] Examples 29 to 37 illustrate the procedures synthetic whereby theintermediate compounds may be prepared to form the second monosaccharide(50).

EXAMPLE 29 1,2:5,6-Di-0-isopropylidene-α-D-glucofuranose (42)

[0246] Sulfuric acid (40 ml) is added dropwise to a solution ofα-D-glucose (41) (50 g, 0,28 mol) in acetone (1 l) at 0° C. The reactionmixture is stirred for 24 h and then it is neutralized using sodiumhydroxide (6 M). The acetone is evaporated and the aqueous layer isextracted with chloroform (×2). The chloroform is dried (MgSO₄) filteredand evaporated. Crystallization from cyclohexane yielded thedi-isopropylidene glucose (42) (41 g, 57%).

EXAMPLE 30 1,2:5,6-Di-0-isopropylidene-3-0-methyl-α-D-glucofuranose (43)

[0247] The α-D-glucofuranose (42) (41 g, 0,16 mol) in tetrahydrofuran(300 ml) is added dropwise to a suspension of sodium hydride (5 g) intetrahydrofuran (200 ml). After 0,5 h, methyl iodide (25 g) intetrahydrofuran (100 ml) is added dropwise to the reaction mixture whichis then stirred for 24 h. Water is added to the reaction mixture whichis then extracted with ether (×3). The ether layer is dried (MgSO₄),filtered and evaporated to yield the methyl protected glucose (43) (38g, 83%).

EXAMPLE 31 3-0-Methyl-αβ-D-glucopyranoside (44)

[0248] The methyl diisopropylidene compound (43) (38 g, 0,14 mol) isdissolved in acetic acid (50%, 700 ml) and the solution refluxed for 18h. After cooling the acetic acid is evaporated. The crude product ispurified by column chromatography eluting withchloroform:methanol:acetone :water (70:27:2:1) to yield3-0-methyl-αβ-glucopyranoside (44) (13 g, 50%).

EXAMPLE 32 Methyl 3-0-methyl-αβ-D-glucopyranoside (45)

[0249] The 3-0-methyl-αβ-glucopyranoside (44) (10 g) is dissolved inmethanol (50 ml) and HCl (conc.) (1 ml) and refluxed overnight. SolidNaHCO₃ is added and the reaction is filtered. The methanol is evaporatedto give 1,3-di-0-methyl-αβ-D-glucopyranoside (45), (95%).

EXAMPLE 33 Methyl4,6-0-benzylidene-3-0-methyl-αβ-glucopyranoside (46)

[0250] The glucopyranoside (45) (8 g) is stirred at room temperature ina solution of benzalaldehyde (20 ml) and zinc chloride (5 g). After 24hours, ice is added and the aqueous layer is extracted with chloroform.The chloroform layer is dried (MgSO₄), filtered and evaporated. Thebenzalaldehyde is removed by vacuum distillation and the product ispurified by silica gel column chromatography eluting withacetone:chloroform (0,5:9,5), to yield benzylidene-αβ-glucopyranoside(46) (60%).

EXAMPLE 34 Methyl4-0-benzoyl-0-methyl-6-deoxy-αβ-glucopyranoside (47)

[0251] The benzylidene compound (46) (5 g) is refluxed at 80° C. in amixture of N-bromosuccinimide (3,7 g) and barium carbonate (4 g) incarbon tetrachloride. After 4 hours, the reaction is filtered and thecarbon tetrachloride is washed with water, dried (MgSO₄), filtered andevaporated to give the bromo compound (70%).

[0252] The bromo compound (4,3 g) is dissolved in a solution of ethanol(300 ml) and nickel chloride (8,6 g) at 0° C. To this solution, sodiumborohydride (8,6 g) in water (50 ml) is added dropwise over a period of15 minutes. The reaction mixture is refluxed at 100° C. for 45 minutes,cooled, filtered and evaporated. Chloroform is added, and the chloroformlayer is washed with water, dried (MgSO₄), filtered and evaporated togive the 6-deoxy sugar (47) (70%).

EXAMPLE 354-0-Benzoyl-3-0-methyl-1-phenylthio-6-deoxy-αβ-glucopyranoside (48)

[0253] The 6-deoxy glucopyranoside (47) (3 g) is dissolved indichloromethane (50 ml). To this solution, phenylthiotrimethylsilane (2g) and trimethylsilyltrifluoromethanesulphonate (0,2 ml) are added. Thesolution is stirred at room temperature overnight, after which saturatedsodium bicarbonate is added. The dichloromethane layer is dried (MgSO₄),filtered and evaporated. The product is purified by silica gel columnchromatography eluting with ethyl acetate:hexane (2:8), to give thecompound (48) (60%).

EXAMPLE 364-0-Benzoyl-3-0-methyl-2-0-pivaloyl-1-phenylthio-6-deoxy-αβ-glucopyranoside(49)

[0254] To a solution of the glucopyranoside (48) (2 g) in pyridine (20ml), pivaloyl chloride (2 ml) is added. The solution is stirred at roomtemperature overnight after which water is added. The aqueous layer isextracted with ethyl acetate, and the organic layer is washed with HCl(6 N). The organic layer is dried (MgSO₄), filtered and evaporated togive the pivaloyl ester (49) (80%).

EXAMPLE 374-0-Benzoyl-3-0-methyl-2-0-pivaloyl-1-fluoro-6-deoxy-β-glucopyranoside(50)

[0255] N-Bromosuccinimide (1,2 g) and diethylaminosulphur trifluoride(1,2 g) are added to a solution of the pivaloyl ester (49) (2 g) indichloromethane (100 ml) at 0° C. After 1 hour, saturated sodiumbicarbonate is added. The dichloromethane layer is dried (MgSO₄),filtered and evaporated. The β-fluoropyranoside (50) is purified bysilica gel column chromatography eluting with ethyl acetate:hexane(2:8), (yield 45%).

[0256] Example 38 illustrates the synthetic procedure whereby thecompound 3-0-(4-0-benzoyl-2-phenylthio-β-D cymaropyranosyl)-12,14β-dihydroxy-pregnan-5-ene-20-one(51) may be prepared.

EXAMPLE 38 3-0- [4-0-benzoyl-2-phenylthio-β-D-cymaropyranosyl]-12,14β-dihydroxy-pregn-5-en-20-one (51)

[0257] Tin chloride (190 mg, 1 mmol) is added to a solution of 3, 12, 14β-trihydroxy pregnan-5-ene-20-one (15) (100 mg, 0,28 mmol) and thefluorocymaropyranoside (38) (210 mg, 0, 56 mmol), in dry diethyl etherand 4 Å molecular sieves at −15° C. The reaction mixture is maintainedat −15° C. for 3 days. Saturated sodium bicarbonate is added to thereaction mixture. The ether layer is dried (MgSO₄), filtered andevaporated. The product is purified by silica gel column chromatographyeluting with chloroform methanol (9, 5:0, 5) to yield the glycoside (51)(30 mg, 15 %).

[0258] Examples 39 to 41 illustrate the synthetic procedures whereby thecymarose and thevetose moieties may be coupled.

EXAMPLE 39 Thevetose-cymarose dissaccharide (53)

[0259] A solution of thevetose (50 A) (1,5 g), cymarose (40) (1,3 g),and molecular sieves 4Å in dichloromethane is stirred at roomtemperature for 1 hour. The reaction mixture is cooled to −15° C., andtin (II) chloride (0,8 g) and silver trifluoromethanesulphonate (1,1 g)are added. The mixture is stirred at −15° C. for 16 hours, after whichtriethylamine (0,5 ml) is added. The reaction product is filtered andthe dichloromethane is evaporated. The dissaccharide (53) is purified bysilica gel column chromatography eluting with ethyl acetate:hexane(2:8), yield 15%.

EXAMPLE 40 Thevetose-cymarose dissaccharide (54)

[0260] To a solution of the dissaccharide (53) (200 mg) intetrahydrofuran (20 ml), tetrabutylammonium fluoride (0,4 ml) is added.The mixture is stirred at room temperature for 1 hour, after whichsaturated sodium bicarbonate is added. The reaction mixture is extractedwith ethyl acetate and the ethyl acetate layer is dried (MgSO₄),filtered and evaporated. The dissaccharide (54) is purified by silicagel column chromatography (acetone:chloroform, 0,5:9,5) yield 60%.

EXAMPLE 41 Thevetose-Cymarose Dissaccharide (55)

[0261] To a solution of the dissaccharide (54) (80 mg) indichloromethane (10 ml), diethylamino sulphur trifluoride (80 μl) isadded at 0° C. After stirring at 0° C. for 0,5 hour, saturated sodiumbicarbonate and more dichloromethane are added. The dichloromethane isdried (MgSO₄), filtered and evaporated. Purification by silica gelcolumn chromatography (ethyl acetate:hexane 1:9), gives thedissaccharide (55) in a 65% yield.

EXAMPLE 42

[0262] The results of the following three bioassays on the appetitesuppressant are set out below, viz.

[0263] a) Irwin Test;

[0264] b) Acute Toxicity Test; and

[0265] c) Oral Dose Anorectic Test.

[0266] a) Irwin Test

[0267] The purpose of this test was to evaluate the appetite suppressantof the invention produced from a plant extract as hereinbeforedescribed, according to the reduced animal Irwin test for tranquillisingand sedative action.

[0268] Experimental Procedure

[0269] The appetite suppressant was extracted from plant material by theApplicant by the method as hereinbefore described and administered totwo of four groups of three animals each: one group receiving notreatment, one group receiving the solvent dimethylsulfoxide (DMSO), onegroup receiving the test sample at 50 mg/kg; and one group receiving thetest sample at 300 mg/kg. Treatment took place by intraperitonealinjection, and observations were made at specific intervals up to fivehours post treatment. Only symptoms other than those observed in theDMSO-treated animals were used in the interpretation of the results.

[0270] Results

[0271] It was clear that the solvent, DMSO, had a marked effect on theanimals, especially on the heat regulating mechanism. Body temperaturesof all the animals treated with the solvent, alone or together with thetest sample, showed a marked drop.

[0272] Animals in the low dose group showed decreased dispersion in thecage and decreased locomotor activity, as in all the other groups,including the control group. Apathy was seen in the same degree as inthe DMSO-treated group. Decreased respiration was observed 15-60 minutesafter treatment. Ptosis (closing of the eyelids) was also observed to alarger degree than in the DMSO group. A pinna (ear) response was seen aswell as a positive finger response, indicating fearfulness. Bodytemperature dropped to 32,7° C. after treatment.

[0273] Animals in the high dose group showed as in the other groups aninitial decreased dispersion in the cage and decreased locomotoractivity, but showed increased dispersion and locomotor activity beforedeath, which occurred approximately 1 hour after treatment. Severeclonic symmetrical convulsions occurred 30 minutes after treatment.Respiration decreased initially, but increased before death. A pinna(ear) response was delayed and a positive finger response was observed,indicating fearfulness, both as observed in animals in the low dosegroup. Body temperature dropped to 30,7° C. after treatment. Increasedpositional passivity was observed as well as decreased body tone.Abnormal limb rotation was observed, the grip strength decreased, nopain response was present and loss of righting reflex occurred.

[0274] Discussion

[0275] When compared with the control and DMSO-treated animals, animalsreceiving the low dose (50 mg/kg) only showed decreased respiration andan increased degree of ptosis. Animals receiving the high dose (300mg/kg) of the test sample reacted very intensely by showing convulsionsand death. All other observations made in these animals can be ascribedto the animals being in convulsions and dying. Signs suggestive oftranquillising and sedative actions such as marked decreased dispersionin the cages, decreased locomotor activity and apathy in the test groupsthat could be ascribed to the test sample were not seen.

[0276] It can therefore be concluded that the test sample is lethal tomice at 300 mg/kg and has respiratory suppressive effects on mice at 50mg/kg, when given intraperitoneally with DMSO as solvent.

[0277] b) Acute Toxicity Test

[0278] The purpose of this test was to gain information on the toxicityof the test sample.

[0279] Experimental Procedure

[0280] A plant extract prepared in accordance with the invention ashereinbefore described, and having appetite suppressive action waspurified and one test sample was tested at increasing doses by oraltreatment in mice. Two animals were used per dose group, except in thehighest dose group where only one animal was treated. Animals wereexamined for good health and their body masses determined on the day oftreatment.

[0281] Doses ranged from 100 mg/kg up to 3 028,5 mg/kg. The dose wascalculated and mixed into prepared potato starch, so that each animalreceived a total dose of 0,2 ml. Animal 13 received 0,25 ml. Potatostarch was prepared by mixing 20 g starch into a small volume of coldwater, and adding it to boiling water, to make up a volume of 1 litre.The suspension was allowed to cool to room temperature before dosing.

[0282] Animals in groups 1 and 2 were treated on the same day. They wereobserved for 24 hours and if no signs of toxicity developed, the nextgroup was treated. The same approach was followed until all the animalswere treated. This schedule was followed to ensure that animals were notunnecessarily treated when an acute toxic dose had been reached in theprevious group.

[0283] Animals were observed for clinical signs of toxicity immediately(1-2 hours) after treatment and daily thereafter. Body mass wasdetermined once a week and total food and water intakes of each animalwere measured.

[0284] Surviving animals were euthanased by intraperitoneal injection ofpentobarbitone sodium (commercially available under the trade nameEuthanaze, Centaur^(R)) on day 14 of the experiment. A post-mortemexamination was performed on these animals, as well as on the one animalwhich died during the experiment. Samples for histopathology werecollected.

[0285] Results

[0286] Group 1 (Control Group)

[0287] No clinical signs of toxicity were observed during the 14-dayobservation period. Food and water intakes were within the normalparameters. Changes in body mass were also within normal parameters. Nohistopathological changes were recorded in the liver samples.

[0288] Group 2 (100 mg/kg)

[0289] No clinical signs of toxicity were observed during theobservation period. Food and water intakes were normal and changes inbody mass over the observation period were also normal. No macroscopicalpathology was observed and no histopathological or morphological changeswere recorded in the liver samples.

[0290] Group 3 (200 mg/kg)

[0291] Animals in this group showed no clinical symptoms of toxicityduring the experiment. Food and water intakes were normal, as was thechange in body mass. No macroscopic pathology was observed, but thelivers showed histopathological changes on examination. Cloudy swellingof the hepatocytes was mild in animal 6, but moderate in animal 5.Moderate hydropic degeneration also occurred in the hepatocytes ofanimal 5.

[0292] Group 4 (400 mg/kg)

[0293] No clinical signs of toxicity were observed during theobservation period, and no macroscopic pathology was observed during thepost-mortem examination. Moderate cloudy swelling and mild hydropicchanges of the hepatocytes were observed on histology.

[0294] Water and food intakes and the increase in body mass in animal 7were normal. Animal 8 consumed almost double the total food intake ofanimal 7 (144,6 g and 73,9 g respectively), but the increase in bodymass was only 0,81 g compared to 2,7 g.

[0295] Group 5 (800 mg/kg)

[0296] One animal (animal 10) died three hours after dosing withoutshowing any specific signs. The other animal (animal 9) survived theentire observation period without any signs of toxicity. Water intake inthe surviving animal was normal (42,42 ml), while food intake was high(134,2 g). The body mass increased by 2,85 g which was the highest ofall animals in the experiment.

[0297] At the post-mortem examination of animal 10, which died shortlyafter oral dosing, the lungs were congested. No foreign body reactionwhich would have indicated inhalation of test material was present. Nomacroscopic pathology was observed in animal 9. Mild cytoplasmicvacuolisation (hydropic degeneration) was present in animal 10, butmoderate in animal 9. The glandular cytoplasmic appearance of the liverwas classified as moderate in both animals.

[0298] Group 6 (1 600 mg/kg)

[0299] None of the animals presented any clinical signs of toxicityduring the duration of the experiment. No macroscopic pathology wasobserved at post-mortem examination, but moderate degenerative changesin the liver of animal 11 were observed at histopathologicalexamination. Animal 12 showed moderate cloudy swelling and mild hydropicchanges of the hepatocytes. Food and water intakes were normal, as wasthe increase in body mass over the experimental period.

[0300] Group 7 (3 028.5 mg/kg)

[0301] Only one animal was treated at this dose. This animal showed nosigns of toxicity during the observation period, and no macroscopicpathology was observed. At histopathological examination, moderatecloudy swelling and hydropic degeneration of the hepatocytes wasobserved. The animal showed a loss of body mass over the observationperiod (−0,82 g), but food and water intakes were normal.

[0302] Discussion

[0303] Since a very small number of animals were used in each dosegroup, it is difficult to make any conclusions. The fact that only oneanimal died at a low dose rate, without showing any symptoms, mightindicate that death was not related to the test sample, but due tostress during and/or after treatment. No animals in higher dose groupsdied or showed any signs of toxicity, which further supports thisassumption.

[0304] The increased food intake observed in animal 8 could possibly beascribed to excessive spillage of food as was reflected in the smallincrease in body mass. It should be kept in mind that all the animals inthis experiment were only treated once, and that it is unlikely that anappetite suppressor will have a marked influence on either the food orwater intakes, or body mass over a 14 day period, as was the case inthis experiment.

[0305] From the histopathological examination of the liver samples, itwas clear that the pathological changes were dose related, with animalsreceiving higher doses showing the extensive changes. The pathologyobserved was not metabolic of nature, but possibly test sample-induced.The changes were only degenerative and therefore reversible. No signs ofirreversible hepatocellular changes were observed.

[0306] It can, therefore, be concluded that only one animal died at alower dose (800 mg/kg), but that the death was possibly not test samplerelated. None of the other animals in any of the dose groups showed anysigns of toxicity during the 14 day observation period after treatment,or died as result of the treatment. A single oral dose of the testsample induced reversible dose-related hepatocellular changes.

[0307] c) Oral Dose Anorectic Test

[0308] The purpose of this test was to determine the activity of a plantextract prepared in accordance with the invention, and the minimumeffective dose, and at the same time investigate any possibleside-effects such as respiratory suppression, as experienced in theIrwin Test (referred to above).

[0309] Experimental Procedure

[0310] Animals were allocated to treatment groups using randomisationtables. Each treatment group consisted of three animals, with 6 animalsin the control group. The test sample was dosed to young female ratswith body weight 100-150 g at acclimatisation, for three consecutivedays. Animals were identified by means of metallic ear tags and KMnO₄skin markings for easy identification. Animals were housed individuallyin standard rodent polycarbonate cages, and water and powderedcommercial rodent pellets were available ad libitum. Water and foodintakes were measured and calculated for each day. In order to find theminimum effective dose of the test sample, five doses were tested.Treatment was by oral gavage, with the test sample suspended in potatostarch.

[0311] The test substance was compound (1), a white granular powderprepared from an extract from plant material in accordance with theinvention, and the measured quantity of the test sample was mixed withprepared potato starch and dosed. Mixing with potato starch took placeimmediately before dosing on each day. Before withdrawal of the dosingvolume for each animal, the suspensions were mixed thoroughly using aVortex.

[0312] A range of five doses was tested, with a control group 15receiving only the carrier substance. Doses were chosen on the basis ofthe effects observed in the aforedescribed Irwin Test and were:

[0313] Group 1: 0,00 mg/kg (Control Group)

[0314] Group 2: 6,25 mg/kg

[0315] Group 3: 12,50 mg/kg

[0316] Group 4: 25,00 mg/kg

[0317] Group 5: 37,50 mg/kg

[0318] Group 6: 50,00 mg/kg

[0319] Results

[0320] Treatment did not affect the health of the animals during thestudy period. Animals treated with the test sample in all dose groups,showed a significantly reduced mean body mass gain over the total studyperiod, and animals in three of the five treatment groups actually lostbody mass.

[0321] Mean food intakes for all the treatment groups were reduced overthe study period. Animals in the higher dose groups showed an increasedwater consumption.

[0322] Respiratory rate in none of the animals in any dose group wassignificantly effected.

[0323] Animals in all dose groups presented with friable livers atpost-mortem examination, but no macroscopic is pathology was observed.

[0324] Discussion

[0325] Data collected during the acclimatisation period confirmed thatall animals included in the experiment were healthy and body mass gainwas comparable between the animals.

[0326] The reduction, and in some animals even a loss, in body massgain, in combination with the reduced food intake is strongly indicativeof suppression of the appetite centre.

[0327] Reduced food intake and reduced body mass gain was experiencedeven with the lowest dose group (6,25 mg/kg). Actual loss in body masswas experienced in the 12,50 mg/kg group.

[0328] It is important to note that the treatment groups all had anincreased water consumption when feed consumption decreased (FIG. 2).This could be due to a diuretic effect of the test sample, or tostimulation of the thirst centre in the brain.

[0329] The fact that no respiratory suppression occurred as had beenobserved in the acute toxicity test referred to above, with theintraperitoneal route, is seen as a positive aspect. This could be dueto reduced absorption from the gastrointestinal tract, with consequentreduced bioavailability. The bioavailability at the oral doses testedwas, however, sufficient for the test sample to be effective. The slightreduction in respiratory rate 1 hour post treatment in most groups couldbe ascribed to filling of the stomach with the dose volume andconsequent passivity of the animals.

[0330] The friable livers observed in the treatment groups could be dueto a change in the energy metabolism secondary to the reduced foodintake, causing increased fat metabolism and overload on the liver. Ifthis was indeed the case, these changes could possibly be regarded thesechanges as transitory which might recover with time after a steady statehad been reached, or after withdrawal of the test sample. The possibleeffect on the liver also needs further investigation.

[0331] Since this study was intended primarily as a screening test,small groups of test animals were used. This makes statisticalinterpretation of the data difficult, especially where individualanimals react totally differently. However, the data indicates that thetest sample has appetite suppressive action, even at the lowest dosetested (6,25 mg/kg). No clinical signs of respiratory suppressionoccurred at the doses tested.

EXAMPLE 43

[0332] Harvested Hoodia plants received either from the naturalenvironment or through a cultivation programme are first stored at 4° C.for a maximum of 48 hours. The plants are washed in tap water andthereafter sliced into ±1 cm slices. The sliced pieces are all combinedand then pressed through a hydraulic press at 300 bar pressure for aminimum of 0.5 hour per pressing. During the pressing the sap of theplant is collected separately. The sap is stored at −18° C. untilfurther processing is required.

[0333] The sap is spray-dried under suitable conditions to obtain a freeflowing powder. The moisture content in the powder is preferably lessthan 5% after spray drying and, if necessary, it is further dried in avacuum oven or using a fluid bed drier.

[0334] Both the sap and the spray-dried material have been showneffective as an appetite suppressant in biological assays in rats.

[0335] Experimental

[0336] 50 kg of Hoodia gordonii plants were washed with tap water andthereafter sliced into 1 cm slices. The sliced plants were then pressedthrough a hydraulic press at 300 bar for a minimum of 0.5 hour perbatch. The sap was collected and the mass was found to be 10 kg whenHoodia gordonii plants from the environment were used, and 20 kg whenHoodia gordonii plants from the cultivation programme was used. The sap(500 g) was spray-dried using the following conditions: Flow rate 2.85ml/min Inlet temperature 110° C. Outlet temperature 70° C. Chambertemperature 78° C.

[0337] The spray-dried powder obtained was a free flowing powder (22 g)with a moisture content of 6.9%.

[0338] The spray dried powder was analysed for active ingredientconcentration using HPLC techniques. The concentration of the active wasdetermined to be 13 g/kg of spray dried powder.

[0339] HPLC Analysis Method Eluant Acetonitrile: water (7:3), isocraticColumn Reverse phase C-18 UV absorbance 225 nm Flow rate 1 ml/minInjection volume 10 μl

[0340] Method

[0341] Spray-dried powder (10 mg) was dissolved in water (0.5 ml) andacetonitrile (0.5 ml) 10 μl of this solution was injected into the HPLCand the concentration of the active compound (1) was determined using astandard curve which was prepared from the pure compound (1).

EXAMPLE 44

[0342] The results of a study designed to assess the possible anorecticeffects of compound (1) in the rat are presented below. In thefollowing, the samples tested are pure sap (Sample 1), spray-dried sap(Sample 2) and active moiety (Sample 3). Samples 1 and 2 are the sap andthe spray-dried sap respectively, as described in Example 43 above.Sample 3 is solvent-extracted compound (1) of ≧95% purity.

[0343] Sample 1 to 3 were each administered as a single oral dose tomale Wistar rats. Two additional control groups received vehicle(distilled water or DMSO). Orally administered fenfluramine (7.5 mg/kg)was included as a reference standard.

[0344] Sample 1 (pure sap) administered orally, produced dose-dependentreductions in food consumption which were statistically significant atdoses of 1600 mg/kg and above when compared with vehicle-treatedcontrols. Concomitant reductions in bodyweight (or growth rate) werealso recorded. On the day of dosing, statistically significant increasesin water consumption were recorded at 3 hours post-dose (6400 and 10000mg/kg) and 6 hours post-dose (10000 mg/kg). Between 24 and 48 hourspost-dose, statistically significant reductions in water consumptionwere recorded at doses of 3200 mg/kg and above.

[0345] Sample 2 (spray-dried sap) administered orally at 76 mg/kg alsoproduced statistically significant reductions in food consumption andbodyweight when compared with vehicle-treated animals. No statisticallysignificant effects on water consumption were recorded.

[0346] Sample 3 (active moiety) produced statistically significantreductions in food consumption at an oral dose of 5.0 mg/kg. Nostatistically significant effects on bodyweights were produced by theactive moiety although examination of the data revealed a slight delayin growth when compared with vehicle-treated control animals. Nostatistically significant effects on water consumption were recorded.

[0347] The reference standard, fenfluramine (7.5 mg/kg), producedstatistically significant reductions in food consumption at 6 and 24hours post-dose when compared with the relevant vehicle-treated controlgroup. No statistically significant effects on water consumption orbodyweight were recorded.

[0348] No treatment-related effects on the livers were recorded. TESTSUBSTANCE Identity Sample 1 (pure sap) Sample 2 (spray-dried sap) Sample3 (active moiety) Appearance Brown liquid Powder White powder Storageconditions −20° C. in the dark Room temperature in the 4° C. in the darkdark Purity Pure sap Pure spray-dried sap ≧95% Vehicle Distilled waterDistilled water Dimethylsulphoxide (DMSO)

[0349] Experimental Procedure

[0350] Fifty-five male Wistar rats were used for the study.

[0351] Bodyweights, food consumption (food hopper weight) and waterconsumption (bottle weight) were recorded daily at the same time eachday from the day of arrival until the termination of the study.

[0352] On Day 1, the rats received a single oral (gavage) dose accordingto the following table: Dose Group n Oral treatment (mg/kg) 1 5 Vehicle(distilled water) — 2 4 Sample 1 (pure sap) 800 3 5 Sample 1 (pure sap)1600 4 5 Sample 1 (pure sap) 3200 5 5 Sample 1 (pure sap) 6400 6 5Sample 1 (pure sap) 10000 7 5 Sample 2 spray-dried sap 38 8 5 Sample 2spray-dried sap 76 9 5 Sample 3 (active moiety) 2.5 10 5 Sample 3(active moiety) 5.0 11 3 Fenfluramine 7.5 12 3 Vehicle (DMSO) —

[0353] Groups 1-8 were dosed using a constant dose volume of 10 ml/kgand groups 9-12 were dosed using a dose volume of 1 ml/kg.

[0354] Food and water consumption were also measured at 1,3 and 6 hoursafter dosing on Day 1.

[0355] Following the measurements of Day 8, the animals were killed bycarbon dioxide asphyxiation, and the livers excised and placed in 10%buffered formalin, prior to histology.

[0356] Paraffin wax sections of each liver were taken at 4-5 μm andstained with haematoxylin and eosin. Additional sections were cut on acryostat at 12 μm and stained for fat with Oil Red O (ORO).

[0357] Data Analysis

[0358] The post-dose food and water consumption measurements andbodyweights at each time-point for the P57-treated animals were comparedwith those for the relevant, similarly-treated vehicle control groupusing analysis of variance followed by Williams' test for comparisonswith controls.

[0359] The data for the fenfluramine-treated animals was compared withthat for the vehicle-treated control group using Student's t test.

[0360] Results

[0361] The results are summarised in the tables.

[0362] Sample 1 (pure sap) administered orally produced marked,dose-related reductions in daily food consumption. The duration andamplitude of these reductions in food consumption were dose-dependent.At 24 hours post-dose, Sample 1 (pure sap) produced statisticallysignificant reductions in food consumption at doses of 1600 mg/kg andabove when compared with vehicle-treated controls. The highest dose ofSample 1 (sap) (10000 mg/kg) produced statistically significantreductions in food consumption on a daily basis up to 5 days post-dose.

[0363] Sample 2 (spray-dried sap) and Sample 3 (active moiety) producedmarked and statistically significant reductions in food consumption atoral doses of 76 and 5.0 mg/kg respectively. In both cases the effectslasted 48 hours post-dose.

[0364] The reference standard, fenfluramine (7.5 mg/kg, p.o.) producedstatistically significant reductions in food consumption at 6 and 24hours post-dose when compared with the relevant vehicle-treated controlgroup (Group 12).

[0365] Sample 2 (spray-dried sap) and Sample 3 (active moiety) producedno marked, dose-related effects on water consumption. On the day ofdosing, the pure sap produced statistically significant increases inwater consumption at 3 hours post-dose (6400 and 10000 mg/kg) and 6hours post-dose (10000 mg/kg). Two days after dosing however,statistically significant decreases in water consumption were recordedin animals receiving Sample 1 (sap) at 3200, 6400 and 10000 mg/kg. Thesereductions however, were not clearly dose-related and only occurredbetween 1 and 2 days post-dose. The biological significance of theseeffects therefore remains unclear.

[0366] Sample 1 (pure sap) produced dose-related, statisticallysignificant effects on bodyweights when compared with thevehicle-treated control group (Group 1). When administered orally atdoses of 3200 mg/kg and above, Sample 1 (pure sap) producedstatistically significant reductions in bodyweight or decreased growthrates when compared with vehicle-treated animals. These effects werestatistically significant from 48 hours post-dose until the end of thestudy.

[0367] Sample 2 (spray-dried sap) administered orally at 76 mg/kg alsoproduced statistically significant reductions in growth of the animalswhen compared with the vehicle-treated control group (Group 1). Theseeffects were statistically significant between Days 3 (48 hourspost-dose) and 5 inclusive.

[0368] Although Sample 3 (active moiety) appeared to delay the growth ofthe animals at the highest dose (5.0 mg/kg) when compared with therelevant vehicle-treated control group (Group 12), this effect was notstatistically significant.

[0369] Fenfluramine, (7.5 mg/kg) produced no marked or statisticallysignificant effects on water consumption or bodyweights when comparedwith the vehicle-treated control group (Group 12).

[0370] No treatment-related effects on the livers were recorded. TABLE1a Effects of oral administration on food consumption in the rat (dailypre-dose data) Group mean food consumption (g ± sd) between Days: GroupOral treatment Dose (mg/kg) −6-−5 −5-−4 −4-−3 −3-−2 −2-−1 1 Vehicle(water) — 27.8 ± 1.54 24.2 ± 1.83 27.6 ± 3.67 28.3 ± 3.50 29.4 ± 2.66 2Sample 1 sap 800 28.3 ± 1.43 24.9 ± 0.82 27.7 ± 0.76 28.4 ± 1.51 30.1 ±0.27 3 Sample 1 sap 1600 29.0 ± 1.39 25.0 ± 2.16 27.4 ± 1.96 28.8 ± 0.6129.5 ± 1.55 4 Sample 1 sap 3200 27.2 ± 2.33 25.1 ± 2.46 26.0 ± 2.52 28.5± 2.29 27.6 ± 1.15 5 Sample 1 sap 6400 28.7 ± 1.64 25.3 ± 1.73 27.3 ±1.45 29.2 ± 1.09 30.3 ± 0.90 6 Sample 1 sap 10000 28.5 ± 2.38 23.7 ±2.73 26.0 ± 2.31 27.0 ± 3.50 28.7 ± 2.26 7 Sample 2 spray-dried 38 28.1± 1.24 23.9 ± 1.79 24.5 ± 2.30 27.6 ± 1.61 28.5 ± 1.87 8 Sample 2spray-dried 76 28.7 ± 0.91 26.5 ± 1.55 27.1 ± 1.01 28.7 ± 1.99 28.9 ±1.37 9 Sample 3 active moiety 2.5 28.8 ± 1.49 26.4 ± 3.12 29.0 ± 1.9929.4 ± 1.76 29.5 ± 2.81 10 Sample 3 active moiety 5.0 28.3 ± 2.1  25.8 ±1.86 28.1 ± 2.65 28.0 ± 2.65 28.5 ± 3.03 11 Fenfluramine 7.5 29.1 ± 0.6625.3 ± 4.03 27.0 ± 1.53 30.8 ± 0.54 29.7 ± 2.84 12 Vehicle (DMSO) — 27.9± 1.8  26.7 ± 2.11 28.7 ± 1.99 28.1 ± 4.06 30.5 ± 2.54

[0371] TABLE 1b Effects of oral administration on food consumption inthe rat (daily post-dose data) Group mean food consumption (g ± sd)between Days: Group Oral treatment Dose (mg/kg) 1-2 2-3 3-4 4-5 1Vehicle (water) — 29.5 ± 3.15 29.6 ± 2.84 30.6 ± 3.49 31.8 ± 3.21 2Sample 1 sap 800 26.1 ± 0.98 29.3 ± 1.49 30.7 ± 1.15 30.9 ± 0.60 3Sample 1 sap 1600  22.6 ± 3.17** 26.9 ± 2.06 30.9 ± 2.54 30.9 ± 1.22 4Sample 1 sap 3200  20.1 ± 1.39**  19.0 ± 1.88**  22.8 ± 1.77** 28.0 ±3.14 5 Sample 1 sap 6400  18.2 ± 4.18**  14.8 ± 1.75**  18.4 ± 0.97** 22.4 ± 3.01** 6 Sample 1 sap 10000  15.1 ± 2.98**  12.4 ± 2.61**  16.0± 3.15**  19.7 ± 4.31** 7 Sample 2 spray-dried 38 25.6 ± 2.85 27.3 ±0.95 30.3 ± 2.06 31.0 ± 2.13 8 Sample 2 spray-dried. 76  24.2 ± 3.25* 25.2 ± 3.24* 29.9 ± 1.85 30.2 ± 2.28 9 Sample 3 active moiety 2.5 26.8± 3.33 29.1 ± 3.43 31.7 ± 3.08 34.0 ± 2.95 10 Sample 3 active moiety 5.0 22.1 ± 2.19††  21.0 ± 3.07†† 27.6 ± 5.26 30.5 ± 3.33 11 Fenfluramine7.5  22.4 ± 3.19^(†) 31.9 ± 0.84 32.7 ± 2.50 33.0 ± 2.55 12 Vehicle(DMSO) — 29.9 ± 3.36 30.6 ± 4.43 30.1 ± 4.17 32.4 ± 5.26 Group mean foodconsumption (g ± sd) between Days: Group Oral treatment Dose (mg/kg) 5-66-7 7-8 1 Vehicle (water) — 30.7 ± 2.24 31.7 ± 3.03 32.9 ± 3.18 2 Sample1 sap 800 33.3 ± 1.69 32.7 ± 0.80  40.1 ± 13.40 3 Sample 1 sap 1600 34.1± 1.36 33.7 ± 1.69 33.8 ± 1.61 4 Sample 1 sap 3200 31.4 ± 2.82 32.3 ±2.91 33.0 ± 3.01 5 Sample 1 sap 6400 26.9 ± 2.81 31.0 ± 2.31 32.0 ± 2.346 Sample 1 sap 10000  22.6 ± 5.70* 30.1 ± 4.79 32.6 ± 5.90 7 Sample 2spray-dried 38 31.8 ± 1.63 31.1 ± 1.94 31.8 ± 2.45 8 Sample 2spray-dried. 76 31.2 ± 2.26 32.3 ± 1.44 33.1 ± 0.61 9 Sample 3 activemoiety 2.5 34.4 ± 4.32 33.1 ± 4.11 34.8 ± 3.71 10 Sample 3 active moiety5.0 33.0 ± 3.16 32.4 ± 3.25 33.0 ± 3.84 11 Fenfluramine 7.5 30.4 ± 0.2332.7 ± 1.90 32.4 ± 1.60 12 Vehicle (DMSO) — 31.8 ± 3.08 32.8 ± 3.98 33.3± 3.76

[0372] TABLE 2a Effects of oral administration on water consumption inthe rat (daily pre-dose data) Group mean water consumption (g ± sd)between Days: Group Oral treatment Dose (mg/kg) −6-−5 −5-−4 −4-−3 −3-−2−2-−1 1 Vehicle (water) — 40.9 ± 4.61 34.8 ± 4.15 37.6 ± 5.63 33.5 ±7.42 32.2 ± 6.32 2 Sample 1 sap 800 38.6 ± 1.96 37.1 ± 9.74 36.4 ± 4.8128.1 ± 1.83 30.4 ± 4.75 3 Sample 1 sap 1600  43.4 ± 10.53 35.9 ± 3.8438.4 ± 4.56 31.1 ± 4.47 36.5 ± 5.39 4 Sample 1 sap 3200 40.1 ± 5.58 33.3± 3.01 37.3 ± 4.46 31.3 ± 3.48 31.7 ± 3.18 5 Sample 1 sap 6400 43.8 ±8.57 36.3 ± 9.02 35.4 ± 8.18 34.0 ± 6.62 35.1 ± 5.72 6 Sample 1 sap10000 37.4 ± 5.34 32.7 ± 3.35 33.2 ± 4.86 29.0 ± 5.11 32.2 ± 3.27 7Sample 2 spray-dried 38 40.0 ± 4.36 35.8 ± 4.92 34.7 ± 3.20 30.2 ± 1.8831.4 ± 2.98 8 Sample 2 spray-dried 76 38.6 ± 1.98 37.0 ± 1.96 48.8 ±21.5 31.6 ± 4.56  39.0 ± 17.27 9 Sample 3 active moiety 2.5 42.0 ± 6.7037.0 ± 5.05 34.1 ± 3.16 28.0 ± 2.58 31.6 ± 3.12 10 Sample 3 activemoiety 5.0 40.9 ± 4.48 34.2 ± 3.00 32.7 ± 1.26 28.2 ± 1.65 33.1 ± 4.8211 Fenfluramlne 7.5 47.0 ± 5.3  35.5 ± 7.49 34.7 ± 3.73 30.9 ± 2.12 31.6± 2.80 12 Vehicle (DMSO) — 43.3 ± 5.67 34.5 ± 4.97 35.2 ± 4.34 28.3 ±4.64 31.4 ± 6.44

[0373] TABLE 2b Effects of oral administration on water consumption inthe rat (daily post-dose data) Group mean water consumption (g ± sd)between Days: Group Oral treatment Dose (mg/kg) 1-2 2-3 3-4 4-5 1Vehicle (water) — 34.9 ± 5.45 36.9 ± 6.06 38.0 ± 7.59 37.2 ± 6.18 2Sample 1 sap 800 30.9 ± 3.77 34.4 ± 8.12  38.2 ± 13.71  35.9 ± 13.51 3Sample 1 sap 1600 29.2 ± 1.66 31.7 ± 5.35  41.3 ± 11.21 34.6 ± 4.10 4Sample 1 sap 3200 35.9 ± 5.88  26.2 ± 2.66* 30.5 ± 2.44 34.1 ± 4.80 5Sample 1 sap 6400  33.4 ± 12.04  27.4 ± 8.13*  32.6 ± 10.67  35.4 ±10.78 6 Sample 1 sap 10000  31.7 ± 12.74  28.5 ± 8.85* 32.4 ± 8.87 36.6± 6.50 7 Sample 2 spray-dried 38 36.0 ± 6.02 34.5 ± 1.79 38.2 ± 7.1639.6 ± 7.09 8 Sample 2 spray-dried 76  45.0 ± 19.03  39.1 ± 16.59  46.9± 18.34 35.9 ± 3.40 9 Sample 3 active moiety 2.5 32.2 ± 4.01  36.1 ±12.42  38.3 ± 11.71  41.5 ± 16.60 10 Sample 3 active moiety 5.0 33.9 ±2.40 31.5 ± 8.12 35.1 ± 3.82 37.7 ± 5.99 11 Fenfluramine 7.5 34.1 ± 3.6037.2 ± 1.48 36.7 ± 3.92 33.8 ± 2.89 12 Vehicle (DMSO) — 40.7 ± 9.10 33.8± 9.37 32.9 ± 7.07  35.2 ± 11.49 Group mean water consumption (g ± sd)between Days: Group Oral treatment Dose (mg/kg) 5-6 6-7 7-8 1 Vehicle(water) — 37.7 ± 5.54 35.3 ± 2.86 36.5 ± 5.85 2 Sample 1 sap 800  39.5 ±11.20 28.8 ± 1.22 31.8 ± 5.58 3 Sample 1 sap 1600  48.1 ± 12.27 37.8 ±7.28 36.9 ± 9.28 4 Sample 1 sap 3200  45.8 ± 18.54  51.0 ± 35.21  42.6 ±13.88 5 Sample 1 sap 6400 45.2 ± 8.72 36.2 ± 6.72 35.9 ± 9.58 6 Sample 1sap 10000  40.7 ± 11.51 38.0 ± 6.66 37.5 ± 6.21 7 Sample 2 spray-dried38 42.7 ± 9.74  45.6 ± 17.15 46.1 ± 9.49 8 Sample 2 spray-dried 76  41.9± 12.37 36.9 ± 8.47 38.1 ± 8.93 9 Sample 3 active moiety 2.5 34.7 ± 7.5733.0 ± 4.20 35.3 ± 8.70 10 Sample 3 active moiety 5.0 39.5 ± 7.78  37.4± 11.07 37.8 ± 6.42 11 Fenfluramine 7.5 33.7 ± 5.43 32.1 ± 1.93 33.6 ±2.50 12 Vehicle (DMSO) — 33.8 ± 9.82 32.3 ± 7.44 32.0 ± 7.22

[0374] TABLE 3a Effects of oral administration on bodyweight in the rat(daily pre-dose data) Group mean bodyweight (g ± sd on Day: Group Oraltreatment Dose (mg/kg) −5 −4 −3 −2 −1 1 Vehicle (water) — 130.9 ± 5.56150.7 ± 5.37 157.3 ± 5.29 168.1 ± 6.20 177.5 ± 6.70 2 Sample 1 sap 800131.6 ± 4.34 150.1 ± 4.84 158.5 ± 4.35 169.6 ± 4.99 177.7 ± 4.10 3Sample 1 sap 1600 130.1 ± 4.3  148.6 ± 6.59 156.7 ± 6.38 167.5 ± 6.04176.6 ± 6.37 4 Sample 1 sap 3200 130.8 ± 6.19 147.7 ± 7.56 154.4 ± 8.06165.2 ± 8.43 175.8 ± 9.10 5 Sample 1 sap 6400 132.6 ± 7.01 151.3 ± 7.23158.4 ± 8.50 169.0 ± 8.79 178.1 ± 7.75 6 Sample 1 sap 10000 132.3 ± 6.75151.8 ± 9.08 157.3 ± 9.37  167.1 ± 10.41  175.4 ± 10.90 7 Sample 2spray-dried 38 131.7 ± 8.28 149.0 ± 5.85 156.2 ± 5.81 166.7 ± 5.54 175.6± 8.42 8 Sample 2 spray-dried 76 130.0 ± 6.99 146.1 ± 6.00 155.9 ± 6.59166.0 ± 6.87 175.1 ± 6.55 9 Sample 3 active moiety 2.5 132.6 ± 7.63148.9 ± 8.51 157.3 ± 8.91 169.8 ± 8.96 179.4 ± 8.71 10 Sample 3 activemoiety 5.0 133.5 ± 6.45 150.5 ± 9.55 158.8 ± 8.48 171.0 ± 7.72 179.0 ±9.20 11 Fenfluramine 7.5 133.2 ± 9.21 152.7 ± 9.09 160.0 ± 9.82 170.0 ±9.15  182.8 ± 10.21 12 Vehicle (DMSO) — 129.1 ± 3.17 147.3 ± 4.37 155.0± 6.29 166.0 ± 5.91 174.8 ± 8.26

[0375] TABLE 3b Effects of oral administration on bodyweight in the rat(daily post-dose data) Group mean bodyweight (g ± sd) on Day: Group Oraltreatment Dose (mg/kg) Pre-dose (1) 2 3 4 5 1 Vehicle (water) — 185.4 ±7.77 192.6 ± 7.16 202.0 ± 10.17 211.2 ± 7.98 220.2 ± 10.35 2 Sample 1sap 800 186.0 ± 4.90 187.0 ± 4.55 198.5 ± 4.20  206.8 ± 5.91 214.8 ±4.65  3 Sample 1 sap 1600 185.0 ± 6.67 186.0 ± 8.28 193.2 ± 6.42  204.0± 6.40 212.4 ± 5.81  4 Sample 1 sap 3200 181.8 ± 9.18 184.6 ± 8.88 186.2± 8.67*  189.8 ± 9.99**  199.2 ± 9.34** 5 Sample 1 sap 6400 186.6 ± 7.96185.6 ± 6.39  183.8 ± 6.87**  185.2 ± 9.18**  191.2 ± 7.89** 6 Sample 1sap 10000  182.8 ± 12.22  181.4 ± 14.06  179.8 ± 15.85**   180.6 ±13.85**  185.6 ± 11.28** 7 Sample 2 spray-dried 38 183.4 ± 8.11 185.8 ±9.23 195.8 ± 7.79  205.6 ± 9.79 214.4 ± 9.61  0 Sample 2 spray-dried 76180.6 ± 6.47 183.4 ± 7.57 188.6 ± 6.73*  198.2 ± 8.50* 206.0 ± 9.43* 9Sample 3 active moiety 2.5 188.2 ± 9.42  191.2 ± 11.15 200.0 ± 11.25 209.6 ± 12.28 219.6 ± 12.95 10 Sample 3 active moiety 5.0  186.4 ±10.02 192.0 ± 9.93 192.4 ± 9.84   201.0 ± 11.27 209.4 ± 12.70 11Fenfluramine 7.5 190.3 ± 9.71  190.3 ± 10.97 197.7 ± 7.37  207.7 ± 7.23217.7 ± 10.69 12 Vehicle (DMSO) — 183.3 ± 8.33  190.3 ± 10.26 199.0 ±10.82  207.7 ± 12.66 215.7 ± 14.05 Group mean bodyweight (g ± sd) onDay: Group Oral treatment Dose (mg/kg) 6 7 8 1 Vehicle (water) — 227.2 ±10.26 235.8 ± 11.82 242.8 ± 11.97 2 Sample 1 sap 800 222.8 ± 4.99  231.5± 3.70  240.0 ± 3.65  3 Sample 1 sap 1600 223.0 ± 6.33  232.6 ± 7.70 240.4 ± 6.66  4 Sample 1 sap 3200  210.6 ± 10.21**  219.0 ± 11.29* 228.4 ± 12.18* 5 Sample 1 sap 6400 201.0 ± 6.89   213.0 ± 6.96**  222.0± 7.94** 6 Sample 1 sap 10000  192.2 ± 10.99** 203.4 ± 11.68  212.4 ±11.35** 7 Sample 2 spray-dried 38 222.6 ± 9.34  231.4 ± 10.62 239.6 ±11.46 0 Sample 2 spray-dried 76 214.0 ± 9.51  222.0 ± 9.49  232.2 ±9.68  9 Sample 3 active moiety 2.5 229.4 ± 13.69 238.4 ± 14.50 247.0 ±14.35 10 Sample 3 active moiety 5.0 219.8 ± 11.86 228.2 ± 12.28 236.0 ±13.95 11 Fenfluramine 7.5 224.3 ± 10.12 234.3 ± 12.70 243.3 ± 9.24  12Vehicle (DMSO) — 222.3 ± 14.84 230.7 ± 15.95 239.0 ± 17.35

[0376] Histopathology Report

[0377] Histological examination was restricted to the liver. Notreatment-related changes were detected for Sample 1 (liquid), Sample 2(spray-dried sap), Sample 3 (active moiety), fenfluramine or the DMSOcontrol group.

[0378] The findings recorded were of a similar incidence in control andtreated groups. TABLE Microscopic pathology incidence summary Group 1Group 2 Group 3 Group 4 Group 5 Group 6 0 800 1600 3200 6400 10000 Sex:Males mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Males on study 5 4 5 5 5 5Animals completed 5 4 5 5 5 5 Liver 5 Examined 5 4 5 5 5 0 Noabnormalities detected 0 0 1 2 3 3 Parenchymal inflammatory cell foci(Total) 0 1 0 0 0 3 Minimal 0 1 0 0 0 1 Hepatocyte hypertrophy -centrilobular (Total) 0 0 0 0 0 1 Minimal 0 0 0 0 0 0 Extramedullaryhaemopoiesis (Total 2 0 0 0 0 0 Minimal 2 0 0 0 0 0 Hepatocytenecrosis - focal (Total) 1 0 0 0 0 0 Minimal 1 0 0 0 0 0 Portal lymphoidinfiltration (Total) 3 4 4 3 2 2 Minimal 3 4 4 3 2 2 Eosinophilichepatocytes - focal (Total) 1 0 0 0 0 0 Minimal 1 0 0 0 0 0 Portalfibrosis (Total) 0 0 1 0 0 0 Minimal 0 0 1 0 0 0 Liver (ORO stain)Examined 5 4 5 5 5 5 No abnormalities detected 2 3 2 4 3 3 Hepatocytefat - centrilobular (Total) 3 1 2 1 2 2 Minimal 3 1 2 1 2 2 Hepatocytefat - periportal (Total) 0 0 1 0 0 0 Minimal 0 0 1 0 0 0 Group 7 Group 8Group 9 Group 10 Group 11 Group 12 38 76 2.5 5 7.5 0 Sex: Males mg/kgmg/kg mg/kg mg/kg mg/kg mg/kg Males on study 5 5 5 5 3 3 Animalscompleted 5 5 5 5 3 3 Liver Examined 5 5 5 5 3 3 No abnormalitiesdetected 2 2 0 1 0 2 Parenchymal inflammatory cell foci (Total) 0 0 0 00 1 Minimal 0 0 0 0 0 1 Hepatocyte necrosis - focal (Total) 0 0 1 0 0 0Minimal 0 0 1 0 0 0 Portal lymphoid infiltration (Total) 3 3 5 4 3 1Minimal 3 3 5 4 3 1 Portal leucocytes (Total) 0 0 1 0 0 0 Minimal 0 0 10 0 0 Liver (ORO stain) Examined 5 5 5 5 3 3 No abnormalities detected 53 3 3 2 2 Hepatocyte fat - centrilobular(Total) 0 2 2 2 1 1 Minimal 0 22 2 1 0

EXAMPLE 45

[0379] A further bioassay, which employed the same test samples asdescribed in Example 44, is described below. Animals in this studyreceived a restricted diet i.e. animals only received food between 12:00and 3:00pm daily. This is different from all other biological assaysconducted thus far, whereby food was available to the rats at lib.Animals were acclimatised over a seven day period (days −7 to −1),dosing took place from day 0 to day 6 at 9:00am by oral gavage. Therecovery period was from days 7 to day 13. Dosage groups are describedin Table 1 below. It should be noted that the actual control group islabelled Group 09. Group 5 is a controlled group which received a dietequivalent to that of Group 4. The purpose of this group was to evaluatethe effect a restricted diet has on the lives of the animals.

[0380] Results

[0381] The results generated during the study showed that theacclimatization period was too short. Rats feed mainly during the nightand the sudden change to a restricted access to feed for 3 hours duringday-time, resulted in low daily intakes. The daily intake of feed wasstill increasing in most groups at the end of the acclimatization periodwhen dosing with the test items started. As a result of this, the effectof the test materials did not significantly affect the food intake ofthe rats during the period of dosing.

[0382] The mean body masses for the different groups for day −7 to −1and days 0 to 6 are shown in the Table D1 and Table D2. The effect ofthe different dosages of the sap and spray-dried sap is shown in theaccompanying graphs as % change in body mass day 0 to 7 (FIG. 5), and %change in body mass day −7 to 7 (FIG. 6). The loss in body mass isclearly dose-related especially with the higher dosages.

[0383] The histopathological examination of the livers did not show anysignificant pathology in the groups receiving the test items.

[0384] Food

[0385] Food consumption was measured daily, during acclimatization andduring the study. Food was available for a 3 hour feeding period daily,starting at 12:00 and ending at 15:00. The animals were fasted for theremainder of the time. Animals in Group 5 received a measured quantityfood on Day 1, equivalent to the average food consumption of Group 4 onDay 0. This controlled feeding pattern for Group 5, as determined fromthe average food consumption of Group 4 from the previous day, wasfollowed for Days 1-7.

[0386] Water

[0387] Water was provided in standard containers. Water (Magalies WaterBoard Tap Water, suitable for human consumption) was available adlibitum. Water consumption was measured once daily, at the same timeeach day, after food consumption determination.

[0388] Acclimatization

[0389] The animals were acclimatized for seven days before the start ofthe study, during which time food and water consumption were determinedas described above. The body masses were determined on a daily basisduring this time. TABLE 1 STUDY DESIGN GROUP TEST NUMBERS DOSE TEST ITEM01 6♂ 001-006 100 mg/kg Frozen sap 02 6♂ 007-012 400 mg/kg Frozen sap 036♂ 013-018 1600 mg/kg Frozen sap 04 6♂ 019-024 3200 mg/kg Frozen sap 056♂ 025-030 CONTROL Elga Option 4 Purified Water 06 6♂ 031-036 2.2 mg/kgSpray-dried sap 07 6♂ 037-042 8.8 mg/kg Spray-dried sap 08 6♂ 043-048 35mg/kg Spray-dried sap 09 6♂ 049-054 CONTROL Elga Option 4 Purified Water

[0390] Route of Administration

[0391] The test items were administered on a daily basis for seven days,using an intra-gastric needle. Animals were fasted for 18 hours prior tothe item administration (starting at 09:00).

[0392] Duration of Treatment

[0393] Animals were treated for seven consecutive days (from Day 0- Day6). Three animals of each group were sacrificed 24 hours after the lastdosing (Day 7). The remaining three animals were sacrificed 7 days afterthe last treatment (Day 13). This procedure was followed for all thegroups except for Group 5 where three animals were sacrificed 24 hoursafter the last controlled feeding (Day 8), the remaining three animalswere sacrificed 7 days after the last treatment (Day 13).

[0394] Body Masses

[0395] Body masses were determined daily, at approximately the same timeeach day for the duration of the study, including during theacclimatization period.

[0396] Euthanasia

[0397] Three animals of each group were sacrificed 24 hours after thelast dosing (Day 7).

[0398] The remaining three animals were sacrificed 7 days after the lasttreatment. This procedure was followed for all the groups except forGroup 5 where three animals were sacrificed 24 hours after the lastcontrolled feeding (Day 8), the remaining three animals were sacrificed7 Days after the last treatment (Day 13). The animals were euthanased atthe end of the study period with CO₂ gas.

[0399] Ophthalmoscopic Examinations

[0400] Ophthalmoscopic examinations, using an ophthalmoscope, were doneprior to the first adminstration of the test item and at termination, inall animals in all groups.

[0401] Macroscopic Pathology

[0402] A full post mortem examination was performed on every animalwhich was euthanased at the end of the study period.

[0403] Histopathology

[0404] Histopathological examination was performed on the liver of eachof the animals. TABLE D.1 MEAN BODY MASSES/GROUP/WEEK Mean body masses(g) & Standard deviation Group Oral treatment Dose (mg/kg) Day −7 Day −6Day −5 Day −4 01 Sample 1 (Sap) 100 203.38 ± 95.39 197.13 ± 90.63 192.75± 89.49 188.62 ± 86.75 02 Sample 1 (Sap) 400 192.53 ± 65.60 183.92 ±61.20 178.25 ± 59.37 173.17 ± 58.10 03 Sample 1 (Sap) 1600 149.25 ±54.80 142.87 ± 51.89 136.85 ± 52.17 132.37 ± 49.64 04 Sample 1 (Sap)3200 224.15 ± 80.70 214.45 ± 77.25 207.10 ± 76.38 201.82 ± 75.42 05 ElgaOption 4 — 214.55 ± 74.90 204.85 ± 72.41 198.57 ± 71.79 193.48 ± 68.49purified water (control) 06 Sample 2 (Spray-dried sap) 2.2 208.65 ±65.74 199.37 ± 62.49 193.18 ± 61.18 188.25 ± 60.89 07 Sample 2(Spray-dried sap) 8.8 256.95 ± 77.55 246.02 ± 73.67 237.47 ± 73.53232.62 ± 71.73 08 Sample 2 (Spray-dried sap) 35 194.37 ± 43.74 185.83 ±42.70 177.53 ± 41.10 172.05 ± 40.13 09 Elga Option 4 — 171.52 ± 69.81162.67 ± 62.68 154.95 ± 61.83 151.38 ± 59.48 purified water (control)Mean body masses (g) & Standard deviation Group Oral treatment Dose(mg/kg) Day −3 Day −2 Day −1 01 Sample 1 (Sap) 100 184.95 ± 84.80 182.48± 83.47 182.25 ± 82.57 02 Sample 1 (Sap) 400 170.82 ± 57.42 168.25 ±58.40 169.37 ± 59.25 03 Sample 1 (Sap) 1600 131.50 ± 49.50 129.67 ±48.89 131.12 ± 48.22 04 Sample 1 (Sap) 3200 198.25 ± 74.82 194.83 ±75.34 196.77 ± 74.56 05 Elga Option 4 — 192.40 ± 67.48 190.87 ± 67.39190.15 ± 65.24 purified water (control) 06 Sample 2 (Spray-dried sap)2.2 186.22 ± 59.98 184.55 ± 58.86 185.97 ± 58.76 07 Sample 2(Spray-dried sap) 8.8 229.78 ± 71.76 228.07 ± 69.88 228.45 ± 68.81 08Sample 2 (Spray-dried sap) 35 170.10 ± 39.49 167.25 ± 37.61 168.00 ±38.83 09 Elga Option 4 — 149.63 ± 57.66 148.30 ± 57.12 149.07 ± 56.01purified water (control)

[0405] TABLE D.2 MEAN BODY MASSES/GROUP/WEEK Mean body masses (g) &Standard deviation Group Oral treatment Dose (mg/kg) Day 0 Day 1 Day 2Day 3 01 Sample 1 (Sap) 100 183.87 ± 83.33 175.83 ± 81.82 175.72 ± 79.05175.48 ± 77.54 02 Sample 1 (Sap) 400 173.45 ± 60.73 164.58 ± 58.52164.75 ± 58.37 166.22 ± 57.69 03 Sample (Sap) 1600 134.38 ± 46.01 129.20± 44.74 127.53 ± 43.20 127.20 ± 41.36 04 Sample (Sap) 3200 199.60 ±75.16 196.38 ± 73.96 192.20 ± 71.20 189.05 ± 69.11 05 Elga Option 4 —194.27 ± 67.46 187.93 ± 65.48 181.97 ± 65.01 177.53 ± 64.73 purifiedwater (control) 06 Sample 2 (Spray-dried sap) 2.2 189.07 ± 60.15 181.52± 58.99 181.48 ± 57.79 184.42 ± 55.64 07 Sample 2 (Spray-dried sap) 8.8230.28 ± 69.32 221.55 ± 68.02 220.17 ± 66.63 221.80 ± 63.88 08 Sample 2(Spray-dried sap) 35 169.10 ± 38.40 164.42 ± 38.03 162.50 ± 36.81 162.75± 36.36 09 Elga Option 4 — 151.02 ± 55.45 146.55 ± 53.77 148.10 ± 52.67149.70 ± 52.05 purified water (control) Mean body masses (g) & Standarddeviation Group Oral treatment Dose (mg/kg) Day 4 Day 5 Day 6 01 Sample1 (Sap) 100 175.53 ± 76.20 177.95 ± 73.99 178.43 ± 72.68 02 Sample 1(Sap) 400 166.55 ± 57.79 169.93 ± 57.47 171.77 ± 57.29 03 Sample (Sap)1600 126.70 ± 39.19 128.00 ± 39.22 128.07 ± 38.66 04 Sample (Sap) 3200186.57 ± 66.29 186.05 ± 67.45 185.68 ± 65.73 05 Elga Option 4 — 174.73 ±61.08 172.85 ± 58.63 171.45 ± 56.79 purified water (control) 06 Sample 2(Spray-dried sap) 2.2 185.75 ± 55.29 189.35 ± 54.66 189.68 ± 53.70 07Sample 2 (Spray-dried sap) 8.8 222.82 ± 63.56 224.82 ± 62.38 224.90 ±62.05 08 Sample 2 (Spray-dried sap) 35 162.52 ± 36.93 164.30 ± 37.69164.22 ± 37.18 09 Elga Option 4 — 152.58 ± 50.37 155.82 ± 49.91 157.85 ±49.70 purified water (control)

[0406] TABLE D.3 MEAN BODY MASSES/GROUP/WEEK (CONTINUED) Dose Mean bodymasses (g) & Standard deviation Group Oral treatment (mg/kg) Day 7 Day 8Day 9 Day 10 01 Sample 1 (Sap) 100 185.38 ± 72.64 234.73 ± 62.44 236.73± 62.39 234.07 ± 62.09 (GHA I 35A) 02 Sample 1 (Sap) 400 178.83 ± 58.24225.63 ± 13.05 277.13 ± 14.18 227.10 ± 14.03 (GHA I 35A) 03 Sample 1(Sap) 1600 132.22 ± 37.08 133.80 ± 55.17 135.23 ± 455.74 134.53 ± 54.96(GHA I 35A) 04 Sample 1 (Sap) 3200 188.57 ± 66.14 199.63 ± 61.07 198.90± 57.48 198.70 ± 54.55 (GHA 9 35A) 05 Elga Option 4 — 173.97 ± 54.29172.98 ± 52.06 157.80 ± 58.62 158.87 ± 57.76 purified water (control) 06Sample 2 (Spray-dried sap) 2.2 196.00 ± 53.09 190.27 ± 27.78 190.27 ±29.54 192.60 ± 29.09 (GHA I 59) 07 Sample 2 (Spray-dried sap) 8.8 231.30± 61.91 177.27 ± 24.48 178.17 ± 23.79 180.67 ± 25.04 (GHA I 59) 08Spray-dried sap 35 167.48 ± 36.75 164.90 ± 22.54 166.63 ± 23.08 168.43 ±22.66 (GHA I 59) 09 Elga Option 4 — 165.50 ± 49.27 193.73 ± 22.37 196.87± 21.86 198.07 ± 21.02 purified water (control) Dose Mean body masses(g) & Standard deviation Group Oral treatment (mg/kg) Day 11 Day 12 Day13 01 Sample 1 (Sap) 100 236.33 ± 62.31 239.07 ± 60.24 238.43 ± 59.85(GHA I 35A) 02 Sample 1 (Sap) 400 229.43 ± 16.97 234.93 ± 18.35 236.20 ±15.97 (GHA I 35A) 03 Sample 1 (Sap) 1600 138.30 ± 53.03 139.30 ± 51.10142.80 ± 49.51 (GHA I 35A) 04 Sample 1 (Sap) 3200 194.73 ± 52.78 194.93± 50.78 197.93 ± 51.57 (GHA 9 35A) 05 Elga Option 4 — 160.80 ± 57.67163.40 ± 56.27 167.80 ± 58.49 purified water (control) 06 Sample 2(Spray-dried sap) 2.2 194.73 ± 29.68 196.97 ± 29.04 198.60 ± 30.18 (GHAI 59) 07 Sample 2 (Spray-dried sap) 8.8 182.03 ± 25.31 185.10 ± 24.60189.73 ± 23.58 (GHA I 59) 08 Spray-dried sap 35 171.67 ± 24.42 174.90 ±25.70 178.57 ± 23.58 (GHA I 59) 09 Elga Option 4 — 199.83 ± 20.21 204.93± 18.65 207.13 ± 18.22 purified water (control)

[0407] TABLE 1 HISTOLOGICAL EVALUATION OF LIVER SECTIONS FROM MALE RATSSample 1 Animal no Hepatic lesions GROUP 1: 100 mg/kg Sample 1 Day 7 01NPL 02 NPL 03 NPL C1 + Day 13 04 NPL MLC 05 FHS1 + 06 NPL GROUP 2: 400mg/kg Sample 1 07 FHS1 + 08 NPL C1 + 09 NPL Day 13 10 DHS1 + 11 NPL 12DHS1 + GROUP 3: 1600 mg/kg Sample 1 Day 7 13 NPL 14 NPL 15 NPL Day 13 16NPL 17 DHS1 + 18 NPL GROUP 4: 3200 mg/kg Sample 1 19 NPL 20 NPL 21 NPLDay 13 22 DHS1 + 23 FHS1 + 24 NPL GROUP 5: CONTROL: ELGA OPTION 4PURIFIED WATER: RESTRICTED FOOD INTAKE GROUP 5: Control: Elga option 4purified water Day 7 25 NPL MLC 26 NPL 27 NPL Day 13 28 DHS1 + 29 DHS1 +30 NPL

[0408] TABLE 2 HISTOLOGICAL EVALUATION OF LIVER SECTIONS FROM MALE RATSSample 2 Animal no Hepatic lesions GROUP 6: 2.2 mg/kg Sample 2 Day 7 31NPL 32 NPL MLC 33 FHS1 + Day 13 34 NPL 35 DHS1 + 36 NPL GROUP 7: 8.8mg/kg Sample 2 37 NPL 38 NPL 39 NPL C1 + Day 13 40 DHS1 + 41 NPL 42 MLCFHS1 + GROUP 8: 35 mg/kg Sample 2 Day 7 43 NPL 44 NPL 45 NPL Day 13 46NPL 47 NPL C1 + 48 MLC FHS1 + GROUP 9: CONTROL: ELGA OPTION 4 PURIFIEDWATER GROUP 9: Control: Elga option 4 purified water Day 7 49 NPL 50 NPL51 FHS1 + Day 13 52 DHS1 + 53 NPL 54 FHS1 +

[0409] No specific lesions were recorded in the liver sections from theexperimental rats which received the frozen sap as well as thespray-dried sap that could be attributed to the oral adminstration ofthe abovementioned chemicals. The hydropic cell swelling recorded inboth control and experimental rats may indicate normal metabolic cellswelling and anoxic changes. Minimal foci of lymphocytic perivascularcuffing were found in some animals and is most likely an incidentalobservation. In a few rats congestion of mild degree is present in thehepatic sinusoids and should be regarded as an incidental observation.

[0410] An important feature of the invention shown by the results ofthis study is that no tolerance to any of the samples developed over thetest period. This may provide considerable benefit, particularly inrelation to the use of the compounds and compositions of the inventionin the treatment of obesity.

[0411] While the compounds and compositions of the invention haveprimarily been described in relation to their properties as appetitesuppressants, it should be noted that this expression—“appetitesuppressant” — is used herein to denote activity which tends to limitappetite and/or increase the sense of satiety, and thus tends to reducetotal calorific intake; this in turn tends to counteract obesity.Accordingly, this invention extends to a method of treating, preventingor combating obesity in a human or non-human animal which comprisesadministering to said human or non-human animal an obesity treating,preventing or combating amount of a compound of formula (2). A preferredembodiment of this aspect of the invention utilises a composition orextract containing a compound of formula (1).

[0412] The term “animal” as used herein extends to, but is notrestricted to, companion animals, e.g. household pets and domesticatedanimals; non-limiting examples of such animals include cattle, sheep,ferrets, swine, camels, horses, poultry, fish, rabbits, goats, dogs andcats.

[0413] As an anorectic agent or in the treatment or prevention ofobesity in a human, a compound of formula (2), preferably of formula(1), or the composition defined in any one of claims 9 and 25-31hereafter, is advantageously administered to said human in a dosageamount of from about 0.01 mg/kg/day to about 10 mg/kg/day. A preferreddosage range is 0.05 mg/kg/day to 0.5 mg/kg/day. When using the spraydried powder form of the extract of this invention, a preferred dosagerange is 0.1 mg/kg/day to 20 mg/kg/day; especially preferred is 0.5mg/kg/day to 5 mg/kg/day.

1-8. Canceled
 9. An extract of a plant of the genus Trichocaulon or thegenus Hoodia, comprising an appetite suppressant agent produced by aprocess including the steps of treating plant material with a solvent toextract a fraction having appetite suppressant activity, separating theextraction solution from the rest of the plant material, removing thesolvent from the extraction solution and recovering the extract.
 10. Acomposition having appetite suppressant activity comprising the extractclaimed in claim
 9. 11. The composition as claimed in claim 10, whereinthe composition is admixed with a pharmaceutical excipient, diluent orcarrier.
 12. The composition as claimed in claim 11, wherein thecomposition is prepared in unit dosage form.
 13. Canceled
 14. Apharmaceutical composition comprising the extract as claimed in claim 9.15. A method of suppressing an appetite by administering to a human oranimal an effective dosage of a composition as claimed in claim 10.16-17 Canceled
 18. An extract of a plant of the genus Trichocaulon orthe genus Hoodia, comprising an appetite suppressant agent produced by aprocess including the steps of pressing plant material to separate sapfrom solid plant material and recovering the sap free of the solid plantmaterial to form the extract.
 19. A composition having appetitesuppressant activity comprising the extract as claimed in claim
 18. 20.The composition as claimed in claim 19, wherein the composition isadmixed with a pharmaceutical excipient, diluent or carrier.
 21. Thecomposition as claimed in claim 20, wherein the composition is preparedin unit dosage form.
 22. Canceled
 23. A pharmaceutical compositioncomprising the extract as claimed in claim
 18. 24. A method ofsuppressing an appetite comprising administering to a human or animal aneffective dosage of the composition as claimed in claim
 19. 25. Amedicament having appetite suppressant activity, said medicamentcomprising the extract as claimed in claim
 9. 26. A medicament havingappetite suppressant activity, said medicament comprising the extract asclaimed in claim
 19. 27-121. Canceled